homologous recombination and human health: the roles of...

Homologous Recombination and HumanHealth: The Roles of BRCA1, BRCA2,and Associated Proteins

Rohit Prakash1,3, Yu Zhang1,3, Weiran Feng1,2,3, and Maria Jasin1,2

1Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York,New York 10065

2Louis V. Gerstner Jr. Graduate School of Biomedical Sciences, Memorial Sloan KetteringCancer Center, New York, New York 10065

Correspondence: [email protected]

Homologous recombination (HR) is a major pathway for the repair of DNA double-strandbreaks in mammalian cells, the defining step of which is homologous strand exchangedirected by the RAD51 protein. The physiological importance of HR is underscored by theobservation of genomic instability in HR-deficient cells and, importantly, the association ofcancer predisposition and developmental defects with mutations in HR genes. The tumorsuppressors BRCA1 and BRCA2, key players at different stages of HR, are frequently mutatedin familial breast and ovarian cancers. Other HR proteins, including PALB2 and RAD51paralogs, have also been identified as tumor suppressors. This review summarizes recentfindings on BRCA1, BRCA2, and associated proteins involved in human disease with anemphasis on their molecular roles and interactions.

Soon after homologous recombination (HR)was discovered to be an important DNA re-

pair mechanism in mammalian cells, an associ-ation between HR deficiency and human dis-ease was uncovered when the hereditary breastcancer suppressors BRCA1 and BRCA2 werefound to be required for HR (Moynahan andJasin 2010; King 2014). Subsequently, germlinemutations in a number of other HR genes havebeen linked to tumor predisposition. Congenitaldefects have also been associated with impairedHR. Tumorigenesis can result from ongoing ge-nomic instability from diminished repair, where-as developmental defects can arise from cell

death/senescence.That HR genes act as genomiccaretakers has generated widespread interest inboth the scientific and medical communities.Because HR defects confer sensitivity to certainDNA-damaging agents, they are being exploitedin cancer therapies. Drugs that cause syntheticlethality in the context of HR defects also holdpromise for treatment (Bryant et al. 2005; Far-mer et al. 2005). This review provides a briefoverview of HR in mammalian cells and sum-marizes the molecular roles of BRCA1, BRCA2,and associated HR proteins involved in humandisease. Extensive discussion of HR pathwayscan be found in Mehta and Haber (2014).

3These authors contributed equally to this article.

Editors: Stephen Kowalczykowski, Neil Hunter, and Wolf-Dietrich Heyer

Additional Perspectives on DNA Recombination available at www.cshperspectives.org

Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a016600

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THE IMPORTANCE OF HR INMAMMALIAN CELLS

DNA lesions, such as double-strand breaks(DSBs), threaten the integrity of the genome,but HR provides a mechanism to precisely re-pair the damage. DSBs repaired by HR are firstend resected to generate 30 single-stranded DNA(ssDNA) (Fig. 1) (see Symington 2014). A DNAstrand-exchange protein—RAD51 in mamma-lian cells—binds to the ssDNA to form a nucle-oprotein filament, which promotes strand inva-sion into a homologous duplex to initiate repairsynthesis (see Morrical 2015). In the synthesis-dependent strand-annealing (SDSA) pathway ofHR, the newly synthesized DNA dissociates toanneal to the other DNA end, and the HR event

is completed by ligation (see Zelensky et al. 2014and Daley et al. 2014). More complex path-ways involve Holliday junction resolution ordissolution (Jasin and Rothstein 2013; see alsoBizard and Hickson 2014; Wyatt and West2014). DSB repair can also occur by a secondmajor mechanism, nonhomologous end join-ing (NHEJ) (Chapman et al. 2012b). NHEJ dif-fers from HR in that the DNA ends are protectedfrom resection before being rejoined; never-theless, deletions and insertions can arise dur-ing NHEJ. The preferred template for HR is theidentical sister chromatid, although the homo-log can be used at lower frequency (Johnson andJasin 2001). The use of the sister chromatid leadsto precise repair, restoring the original sequencethat was present before damage, but it is limited

DSB

End resection

RAD51 loadingStrand invasion

NHEJ

Repair synthesis

HR

Δ, +

End protection

Figure 1. Simplified schemes of double-strand break (DSB) repair by homologous recombination (HR) andnonhomologous end joining (NHEJ). Once a DSB is generated, it can be processed for HR by end resectionproteins, leading to ssDNA. The RAD51 strand-exchange protein forms a nucleoprotein filament with ssDNAthat invades an unbroken homologous DNA, typically the sister chromatid, as shown. The 30 end primes DNAsynthesis from the homologous DNA; using the sister chromatid, the repair can be precise to restore the originalsequence before damage. To complete HR, the newly synthesized strand can dissociate to anneal to the other end.Other outcomes are also possible, for example, in which Holliday junctions are formed and either dissolved orresolved. Alternatively, DNA ends are protected from end resection by NHEJ proteins; subsequent steps in NHEJcan result in mutagenic repair with deletions and insertions (D, þ).

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to the S/G2 phases of the cell cycle, whereasNHEJ is operational throughout the cell cycle(Rothkamm et al. 2003).

Although HR has long been known to be amajor DNA repair mechanism in bacteria andyeast (see Mehta and Haber 2014; Reams andRoth 2015), the importance of HR in the main-tenance of mammalian genome integrity hasonly emerged in the last two decades. Directevidence came from molecular analysis of DSBrepair, in which HR and NHEJ are both foundto be robust repair mechanisms (Fig. 1) (Rouetet al. 1994; Liang et al. 1998; Johnson and Jasin2000). This finding forms the basis of currentgenome-editing approaches in mammalian cells(Cong et al. 2013; Mali et al. 2013).

Strong genetic evidence for the importanceof HR comes from the study of mice deficientin the RAD51 strand-exchange protein. Rad51

disruption is lethal early in embryogenesis andRad51 null cells cannot be propagated (Limand Hasty 1996; Tsuzuki et al. 1996). The lethal-ity is attributed to the impaired repair of lesionsthat arise during DNA replication in the rapidlycycling cells of the embryo. Thus, a critical func-tion of HR is likely to be the repair of replica-tion-associated damage (see Syeda et al. 2014),as is the case in bacteria (Cox et al. 2000). HR isalso critical for the repair of interstrand cross-links (Long et al. 2011).

HR and NHEJ can “compete” for the re-pair of the same lesion but also “collaborate”in the repair of distinct lesions (Fig. 2A) (Kassand Jasin 2010). Competition is evidenced in astandard HR reporter assay in which a DSB isinduced by the I-SceI endonuclease: HR, mea-sured as GFPþ cells, is elevated in NHEJ mutantcell lines relative to wild-type cells (Fig. 2B)

CompetitionDSB

Collaboration

I-Scel

HR

TUNEL/DAP1/Tuj1

Postmitotic layerProliferating layer

SVl.

ll. lV.

lll.

SVSV

Xrcc2–/–

Lig4–/–

VZ

VZ

VZ

GFP+

FL2 FL2

FL1

Wild type2%

Ku70–/–

11%

NHEJ

HRA

B C Wild type

Figure 2. Competition and collaboration between double-strand break (DSB) repair pathways. (A) Schematic ofthe interactions between the homologous recombination (HR) and nonhomologous end joining (NHEJ)pathways for DSB repair. (B) Competition for repair of a DSB. In the direct repeat green fluorescent protein(DR-GFP) reporter assay, a DSB repaired through HR restores a functional GFP gene, as detected by flowcytometry. NHEJ-deficient Ku702/2 cells show substantially elevated HR, showing how HR and NHEJ canact on the same DSB, such that NHEJ suppresses HR (modified from Pierce et al. 2001). The DSB is generated byI-SceI endonuclease. (C) Collaboration between HR and NHEJ, as illustrated in the embryonic brain. (i,ii)Apoptosis is rare in the wild-type embryonic brain (E13.5), as indicated by the lack of TdT-mediated dUTP-biotin nick end labeling (TUNEL) staining in either the proliferating ventricular zone (VZ) or the postmitoticsubventricular (SV) zone (marked with Tuj1). (iii) HR-deficient Xrcc22/2 cells show substantial apoptosis in theVZ. (iv) In contrast, NHEJ-deficient Lig42/2 cells predominantly have elevated apoptosis in the SV. Therefore,HR and NHEJ both contribute to the integrity of the embryonic brain (modified from Orii et al. 2006).

HR Proteins Linked to Human Disease

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(Pierce et al. 2001). The rescue of certain HRmutants with combined NHEJ deficiency alsospeaks to the competition between pathways(see below) (Bouwman et al. 2010; Buntinget al. 2010). Collaboration between HR andNHEJ is illustrated in the different cell layersof the embryonic brain. HR is required in theproliferating cell layer and NHEJ is required inthe postmitotic cell layer, such that mutation ofeither an HR or NHEJ pathway componentleads to high levels of apoptosis but in a distinctcell layer (Fig. 2C) (Orii et al. 2006). Collabora-tion between pathways is also observed in someHR/NHEJ double mutant mice, which showmore severe phenotypes than either single mu-tant (Couedel et al. 2004; Mills et al. 2004). HRand NHEJ can even be used to repair the samelesion through a break-induced replication-type event, which is initiated by HR and com-pleted by NHEJ (Richardson and Jasin 2000).Special cases are the programmed DSBs, whichare channeled into defined repair pathways, forexample, HR for SPO11-generated DSBs duringmeiosis (see Lam and Keeney 2015) and NHEJfor RAG-induced DSBs in the immune system(Chapman et al. 2012b).

Early studies of mammalian homologs ofyeast HR genes did not link HR to tumor sup-pression. Mouse knockouts showed embryoniclethality (Rad51, Lim and Hasty 1996; Tsuzukiet al. 1996) or, at the other extreme, little or nophenotype (Rad52, Rijkers et al. 1998; Rad54,Essers et al. 1997). However, characterization ofBRCA1 and BRCA2 led to the discovery of thelink between HR and human health, in partic-ular, tumor suppression (Moynahan and Jasin2010). Subsequent work has implicated a num-ber of other HR proteins, including PALB2 andthe RAD51 paralogs.

BRCA1

BRCA1 Mutations in Patients

Breast cancer early onset gene 1 (BRCA1) wasidentified in the early 1990s as one of the majorhereditary breast cancer susceptibility genes(Futreal et al. 1994; Miki et al. 1994; King2014). Germline mutations in BRCA1 confer ahigh lifetime risk for breast (�60%) and ovar-

ian (�40%) cancer (average cumulative risks byage 70), as well as a lesser increase in risk forpancreatic, prostate, and other cancers (Kinget al. 2003; Metcalfe et al. 2010). Mutation car-riers are heterozygous, whereas tumors oftenshow loss of the wild-type allele (see below)(Futreal et al. 1994). BRCA1-mutated breastcancers are typically basal-like rather than lumi-nal, and negative for estrogen and progesteronereceptors and human epidermal growth factorreceptor 2 (HER2) amplification (i.e., “triplenegative”); thus, they do not respond to hor-monal therapies or therapies that target HER2,making them particularly difficult to treat(Foulkes et al. 2003).

BRCA1 mutant allele frequencies are suffi-ciently high that individuals with biallelic mu-tations could be expected in the population;however, until recently, only one individualwith bona fide deleterious mutations in bothBRCA1 alleles has thus far been reported (Dom-chek et al. 2013), consistent with the embryoniclethality associated with BRCA1 loss in mice(Moynahan 2002). This individual likely sur-vived to adulthood because one of her BRCA1alleles is hypomorphic. However, she had de-velopmental issues, early onset ovarian cancer,and toxicity from carboplatin and paclitaxeltherapy (Domchek et al. 2013). The identifica-tion of an individual with biallelic BRCA1 mu-tations has important implications because thecombination of congenital issues, cancer, andsensitivity to interstrand cross-linking agentslike platinum-based drugs is associated with abroadly defined syndrome called Fanconi ane-mia (D’Andrea 2013). More recently, a secondindividual with biallelic BRCA1 mutations hasbeen identified, in this case, with breast canceras well as congenital abnormalities (Sawyer et al.2015). Thus, biallelic mutations in BRCA1 arenow considered to cause a distinct subtype ofFanconi anemia (FA-S).

BRCA1 Domains and Interactions

Human BRCA1 encodes an 1863 amino acidprotein that can be divided into three regions,the amino terminal Really Interesting New Gene(RING) domain, a central part with a large un-

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structured region encoded by exon 11 followedby a coiled-coil domain, and tandem BRCA1carboxy-terminal repeats (BRCTs) (Fig. 3) (Liand Greenberg 2012). Through these domains,BRCA1 forms complexes with several proteins,implicating BRCA1 in multiple cellular func-tions, such as transcription regulation, cell-cyclecheckpoint activation, and DNA repair (Venki-taraman 2014). Importantly, the role of BRCA1in DNA repair, in particular in HR repair (Moy-nahan et al. 1999), has thus far been one of itsmost well-recognized functions thought to becritical for its tumor suppressor activity.

RING Domain Interactions with BARD1

BRCA1 forms a heterodimer with BARD1, an-other RING and BRCT-containing protein,through helices flanking the core RING motif(Fig. 3) (Wu et al. 1996; Brzovic et al. 2001).BARD1 appears to be an obligate partner ofBRCA1 because BRCA1-BARD1 interaction isessential for their mutual stability in vivo, and

Brca1 and Bard1 knockout mice show identicalphenotypes (McCarthy et al. 2003; Shakya et al.2008). Although not nearly as prevalent asBRCA1 mutations, germline BARD1 mutationshave also been reported in breast and ovariancancer families (De Brakeleer et al. 2010; Saba-tier et al. 2010; Ratajska et al. 2012).

As a RING domain is a common proteinstructure for E3 ubiquitin ligases, the E3 ligaseactivity of the BRCA1-BARD1 heterodimer hasbeen a focus of interest. BRCA1-BARD1 candirect both mono- and polyubiquitylation de-pending on the E2 conjugating enzyme (Chris-tensen et al. 2007). In addition to more standardlinkages, BRCA1-BARD1 catalyzes the forma-tion of noncanonical lysine6-linked ubiquitinchains (K6-polyUb), which likely serve as a sig-nal for complex assembly and/or protein stabi-lization rather than degradation (Wu-Baer et al.2003; Nishikawa et al. 2004). In vivo, BRCA1-BARD1 appears to play a critical role in theaccumulation of K6-polyUb conjugates atDSBs (Morris and Solomon 2004). Substrates

BRCA1

BARD1

PALB2 RAD51

RAD51 paralogs

B C

C

D X2

X3

BRCA2

DSS1

RING BRCT

WD40

BRC repeatsDNA-bindingdomain

C-ter

BRCT

pSXXF pSXXF BRCA1complexprotein

Abraxas

BRIP1CtIP

A

BC

Coiled-coil

Coiled-coil

RING

Figure 3. Protein interactions for a functional homologous recombination (HR) pathway. BRCA1 executes itsvarious functions using the RING, coiled-coil, and BRCT domains. BRCA1 and BARD1 interact at theirrespective RING domains. BRCA1 BRCT motif mediates interaction through a pSXXF motif in Abraxas,BRIP1, and CtIP in the A, B, and C complexes, respectively. The coiled-coil domain of BRCA1 recognizes thecoiled-coil domain of PALB2, which, in turn, binds BRCA2 through its WD40 domain. BRCA2 has mediatoractivity for loading RAD51 onto replication protein A (RPA)-coated ssDNA (BRC repeats) and for the stabi-lization of the RAD51 presynaptic filament carboxy terminal (C-ter) domain. RAD51 paralog complexes alsointeract with RAD51 and may promote/stabilize RAD51 filaments. BRCA2 also has a DSS1 and DNA-bindingdomain that, although not required for HR, likely is required for optimal HR levels. This domain consists of fourglobular domains and a helical tower domain with a three-helix bundle at its apex.





for BRCA1-BARD1-mediated polyubiquityla-tion include BRCA1 itself and the end resectionfactor CtIP (Yu et al. 2006), and, for mono-ubiquitylation, include histone H2A, whichhas been implicated in the maintenance of si-lenced heterochromatin (Zhu et al. 2011).

The in vivo significance of the BRCA1 E3ligase activity to tumor suppression has beenchallenged by recent reports showing that theBARD1-interaction-proficient but E3 ligase-deficient BRCA1 I26A mouse mutant is compe-tent for HR and is not tumor prone (Reid et al.2008; Shakya et al. 2011). In contrast, the can-cer-associated C61G RING domain mutant,which disrupts the interaction with BARD1rather than specifically impairing interactionwith E2 enzymes, causes tumor susceptibilityin mice as well as in patients (Wu et al. 1996;Drost et al. 2011). Another deleterious mutant,BRCA1 C64R, also has impaired interactionwith BARD1 (Caleca et al. 2014). Thus, whereasthe integrity of the BRCA1 RING domain re-gion is critical for tumor suppression, appar-ently because of its interaction with BARD1,the physiological role of the E3 ligase activityitself is uncertain. One possibility is that auto-ubiquitylation of BRCA1 increases its stabil-ity because the BRCA1 I26A mutant protein ispresent at lower levels in cells (Reid et al. 2008).These cells have elevated DNA-damage-in-duced genomic instability, suggesting the pos-sibility that wild-type levels of BRCA1 are re-quired for maintenance of genomic integrity.

Coiled-Coil Domain Interaction with PALB2

Through its coiled-coil domain, BRCA1 inter-acts with the bridging protein PALB2, whichconnects BRCA1 with the other major heredi-tary breast cancer suppressor BRCA2 (Fig. 3)(Xia et al. 2006; Sy et al. 2009; Zhang et al.2009a,b). BRCA1-PALB2-BRCA2 interactionplays an important role in RAD51 cellular dy-namics and will be discussed further below.

BRCT Interactions with Several Proteins

The BRCTrepeats mediate interactions betweenBRCA1 and several proteins involved in the

DNA-damage response, including Abraxas/FAM175A, BRIP1/BACH1, and CtIP/RBBP8,which are part of the BRCA1-A, -B, -C complex-es, respectively (Moynahan and Jasin 2010; Liand Greenberg 2012). The BRCA1 BRCTrepeatsrecognize a phosphorylated serine in a pSXXFmotif (Yu et al. 2003b), which exists in each ofAbraxas, BRIP1, and CtIP (Fig. 3). Importantly,BRCT repeats on one BRCA1 molecule can beoccupied by only one pSXXF motif, implyingthe mutually exclusive composition of theBRCA1-A, -B, and -C complexes. The impor-tance of the interaction between BRCA1 andphosphorylated proteins at the BRCT repeatsis emphasized by the findings that disruptionof this interaction is associated with tumorsusceptibility in mice and humans as well aswith reduced HR (Shakya et al. 2011). A chal-lenge is to determine which of the multiple pro-tein interactions is/are critical for tumor sup-pression.

The BRCA1-A complex targets BRCA1 toubiquitin conjugates at DSBs, which are criticalfor DNA-damage signaling and repair (Huenet al. 2007; Doil et al. 2009). Deficiency ofUBC13, the E2 enzyme required for these ubiq-uitin conjugates, causes severe defects in DNA-damage signaling and HR (Wang and Elledge2007; Zhao et al. 2007). In addition to BRCA1,Abraxas binds to another component of theBRCA1-A complex RAP80, which recognizespolyubiquitylated histones like H2AX to recruitthe BRCA1-A complex to DNA-damage sites(Kim et al. 2007; Sobhian et al. 2007; Wanget al. 2007). The BRCA1-A complex consistsof other proteins with ubiquitin-binding do-mains and also with deubiquitylation activity,which can provide complex regulation of pro-tein dynamics at damage sites (Huen et al. 2010).Notably, BRCA1-A and -C complexes appear tohave opposite roles in an early step of HR, DNAend resection (discussed below).

The BRIP1 component of the BRCA1-Bcomplex is a helicase that unwinds secondaryDNA structures, such as four-stranded struc-tures (G4 DNA), which may impede DNA rep-lication (Cantor et al. 2004; London et al.2008; Wu et al. 2008). BRCA1 recruits phos-phorylated BRIP1 to chromatin during S phase,

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and the BRCA1-B complex is required forS-phase checkpoint activation when replica-tion forks are stalled or collapsed (Cantoret al. 2001; Litman et al. 2005; Greenberg et al.2006). Biallelic BRIP1 mutations, which causecellular sensitivity to cross-linking agents, havebeen identified in patients with developmen-tal issues and cancer predisposition, such thatBRIP1 is considered to be a Fanconi anemiagene subtype (FA-J); monoallelic mutationsare associated with breast and ovarian cancerpredisposition (Cantor et al. 2001; Levituset al. 2005; Levran et al. 2005; Litman et al.2005; Seal et al. 2006; Rafnar et al. 2011).

BRCA1 Function in HR

A link between BRCA1 and RAD51 came fromthe observation of their subcellular colocaliza-tion in nuclear foci (Scully et al. 1997). An es-sential role for BRCA1 in HR was established bythe direct demonstration of substantially re-duced HR in BRCA1 mutant cells (Moynahanet al. 1999, 2001a). These studies further showedthat BRCA1 mutant cells follow the paradigm ofother HR mutant mammalian cells identifiedaround the same time, in terms of having spon-taneous chromosome instability and high sen-sitivity to cross-linking agents (Moynahan et al.2001a). Multiple breast cancer and engineeredmutations in BRCA1 have been shown to conferdefects in HR, connecting the HR and cancersuppressor roles of BRCA1 (Ruffner et al. 2001;Sy et al. 2009; Drost et al. 2011; Towler et al.2013). BARD1 has also been shown to be im-portant for HR (Westermark et al. 2003; Lauferet al. 2007). BRCA1-deficient cells are exquisite-ly sensitive to inhibitors of poly(ADP-ribose)polymerases (PARP) (Bryant et al. 2005; Farmeret al. 2005; McCabe et al. 2006), which functionin DNA single-strand break repair. Syntheticlethality between PARP inhibition and HR de-ficiency is currently being explored as an ap-proach to cancer therapy.

The molecular mechanism by whichBRCA1 contributes to HR has been extensivelystudied in the past decade. Compelling evi-dence suggests that BRCA1 functions in twodistinct steps: (1) 50 to 30 resection of DSBs to

generate 30 ssDNA overhangs, and (2) loading ofthe RAD51 recombinase onto the ssDNA.

BRCA1 and End Resection

The involvement in resection was first suggestedby the observation that BRCA1 mutant cells aredefective in a second homology-based DSB re-pair pathway, single-strand annealing (SSA),which, like HR, relies on a resection intermedi-ate but diverges at later steps (Fig. 4) (Stark et al.2004). End resection is a key step in DSB repairpathway choice, promoting pathways that usehomology while suppressing canonical NHEJ(Kass and Jasin 2010). Consistent with a rolein resection, BRCA1 colocalizes with the resec-tion complex MRE11-RAD50-NBS1 (MRN) af-ter DNA damage and directly interacts with theresection factor CtIP (Wong et al. 1998; Yu et al.1998; Zhong et al. 1999; Sartori et al. 2007).

Onemodel isthatBRCA1interactswithphos-phorylated CtIP (BRCA1-C complex) throughits carboxy-terminal BRCT domain to cooperatewith the MRN nuclease to catalyze resection(Wong et al. 1998; Yu et al. 1998; Sartori et al.2007; Chen et al. 2008). As CDK-dependentphosphorylation of CtIP is required for CtIP ac-tivation and BRCA1-CtIP interaction, it has beenproposed that BRCA1 promotes resection byrecruiting CDK-phosphorylated/activated CtIPto DSB sites (Yu et al. 2003b; Yu and Chen 2004;Yun and Hiom 2009; Buis et al. 2012). Surpris-ingly, however, a CtIPmouse mutant defective forBRCA1 interaction has recently been reported tosupport HR to a similar extent as wild-type CtIP(Reczek et al. 2013), questioning the biologicalsignificance of the BRCA1-phospho-CtIP inter-action. Possibly, other proteins target CtIP todamage sites (Daugaard et al. 2012).

In addition to specifically promoting resec-tion, BRCA1 also appears to act as an antago-nizer of the resection suppressor 53BP1 (Fig. 4)(Bouwman et al. 2010; Bunting et al. 2010).In the absence of BRCA1, 53BP1 accumulatesat DSBs to block resection and HR, ultimatelyleading to chromosomal aberrations and celldeath; deletion of 53BP1, however, rescues theviability of BRCA1 mutant cells and mice, as





well as the HR defects of BRCA1-deficient cells.Analysis of BRCA1 and 53BP1 in DNA-dam-age-induced foci by superresolution micro-scopy suggests that BRCA1 spatially excludes53BP1 from the proximity of DSBs during Sphase (Chapman et al. 2012a).

As CtIP-dependent resection is proficientwith the combined absence of BRCA1 and53BP1 (Bunting et al. 2010), it appears that therole of BRCA1 in relieving 53BP1 is upstreamof recruiting CtIP at DSBs. The balance be-tween BRCA1 and 53BP1 at DSBs has beenshown to be regulated by acetylation of histoneH4K20me2, which interferes with 53BP1 bind-ing (Tang et al. 2013) and also involves RIF1,which binds ATM-phosphorylated 53BP1(Chapman et al. 2013; Escribano-Diaz et al.2013; Feng et al. 2013; Zimmermann et al.2013). The BRCA1-RIF1 antagonism in humancells has been reported to involve BRCA1 inter-action with phosphorylated CtIP (Escribano-

Diaz et al. 2013), although it is uncertain as yethow to reconcile this finding with the lack of anHR phenotype in the phosphor-CtIP mutantmouse cells (Reczek et al. 2013).

Surprisingly, an antiresection activity ofBRCA1-A complex members has also been re-ported (Fig. 4). Depleting members like RAP80results in a hyperrecombination phenotype as-sociated with increased CtIP-dependent resec-tion (Coleman and Greenberg 2011; Deveret al. 2011; Hu et al. 2011; Kakarougkas et al.2013). The RAP80 subunit of the BRCA1-Acomplex has been suggested to inhibit resectionby binding to ubiquitin chains at DSBs (Biswaset al. 2011; Coleman and Greenberg 2011); itsinhibitory effect can be relieved by the de-ubiquitylating factor POH1 (Kakarougkas et al.2013). Based on the mutual exclusiveness ofthe BRCA1 complexes, a model has been pro-posed to explain this observation, in which theabsence of the BRCA1-A complex allows more

DSB

Endresection

53BP1

BRCA1

BRCA1-A

PALB2BRCA2

RAD51

HR

SSA

Strandannealing

Repeats present

BRCA1-C

BRCA1

Figure 4. BRCA1 and BRCA2 have distinct roles in HR. BRCA1 acts at an early HR step to promote end resectionand at a later step to recruit PALB2 and, hence, promote BRCA2 chromatin localization. BRCA1 acts by antag-onizing the resection inhibitor 53BP1. It may further regulate resection by recruiting CtIP (gold ball) in theBRCA1-C complex, while inhibiting end resection in the BRCA1-A complex containing Abraxas and RAP80(black and gray balls); alternatively, Abraxas-RAP80 may act independently of BRCA1 to suppress resection.BRCA2 promotes loading of RAD51 recombinase onto the resection product to form an RAD51-ssDNA fila-ment, which is essential for HR and prevents the engagement of the 30-ssDNA into the deleterious single-strandannealing (SSA) pathway. SSA acts when homologous repeats are present and leads to a deletion of sequencesbetween the repeats.

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BRCA1 to functionally interact in other com-plexes that promote resection (Coleman andGreenberg 2011; Hu et al. 2011). Physiological-ly, the promotion and inhibition of end resec-tion by different complexes can act to fine tunethe response. The opposing roles of BRCA1complexes may help to explain the different de-grees of resection defects upon BRCA1 disrup-tion reported in the literature (Chen et al. 2008;Kakarougkas et al. 2013; Zhou et al. 2013).

BRCA1 and RAD51 Loading

In addition to its role in end resection, BRCA1appears to have a downstream role in HR bypromoting the localization of downstream HRfactors (Fig. 4). Resected DNA is a substrate forRAD51 binding, but it is initially bound by thessDNA-binding replication protein A (RPA), re-quiring mediator proteins like BRCA2 to assistRAD51 loading onto ssDNA concomitant withRPA eviction (see Zelensky et al. 2014). BRCA1promotes the recruitment of BRCA2 to DSBsthrough the bridging protein PALB2 (Fig. 3)(Xia et al. 2006; Sy et al. 2009; Zhang et al.2009a,b). The anchor role of BRCA1 is indi-cated by the hierarchy of the DNA-damage-induced focus formation: BRCA1 disruptiondiminishes PALB2, BRCA2, and RAD51 foci;PALB2 disruption reduces BRCA2 and RAD51foci but not BRCA1 foci; BRCA2 disruptiononly impairs RAD51 foci. Consistent withthe idea that BRCA1 facilitates RAD51-depen-dent HR through PALB2, clinical BRCA1 mu-tations, which abrogate BRCA1-PALB2 inter-actions, cause HR defects. The role of BRCA1in RAD51 loading appears to be dispensablewhen 53BP1 is absent, at least in mouse cells,however, as eliminating 53BP1 rescues the HRdeficiency of BRCA1 null cells (Bouwman et al.2010; Bunting et al. 2010,2012). It seems possi-ble that 53BP1 loss creates a favorable conditionfor RAD51 loading (e.g., creating hyperresectedends), which bypasses a downstream role forBRCA1 in HR.

The BRCA1 coiled-coil domain, whichbinds PALB2, is distinct from the BRCT domain(Fig. 3). However, mutations in the BRCT do-main often result in destabilization of the mu-

tant BRCA1 protein (Williams and Glover 2003;Williams et al. 2003; Lee et al. 2010) and thusimpair RAD51 loading despite an intact PALB2-interaction domain (Johnson et al. 2013). Inter-estingly, therapy-resistant human breast cancercells have been isolated in which HSP90 stabili-zation of the mutant BRCA1 protein, combinedwith reduced 53BP1 levels, restores RAD51 fo-cus formation (Johnson et al. 2013). These re-sults emphasize the complexity of acquired re-sistance involving BRCA1.

BRCA2

BRCA2 Mutations in Patients

BRCA2 is the second major hereditary breastcancer susceptibility gene (Wooster et al. 1995;Tavtigian et al. 1996). BRCA2 breast cancers aredistinct from those with BRCA1 mutations inthat they are generally of the luminal subtype,rather than basal-like, and so are often estro-gen-receptor positive (Jonsson et al. 2010; Wad-dell et al. 2010). The difference in breast cancertype may be related to the observation thatBRCA1 mutations alter mammary progenitorcell fate commitment (see Proia et al. 2011,and references therein). Like BRCA1, BRCA2mutation carriers are also predisposed to ovari-an cancer as well as other tumors at a lower pen-etrance, such as that of the prostate and pancreas(Ozcelik et al. 1997; Consortium 1999; Anto-niou et al. 2003; Edwards et al. 2003; King et al.2003; van Asperen et al. 2005). DeleteriousBRCA2 mutations, as with BRCA1, have beenreported throughout the length of the protein,mostly truncating mutations but also pointmutations (Breast Cancer Information Core,research.nhgri.nih.gov/bic/). Mutations of un-certain clinical significance have also been iden-tified such that several approaches have been de-vised to evaluate their functional significance(Hucl et al. 2008; Kuznetsov et al. 2008; Changet al. 2009; Biswas et al. 2011, 2012; Bouwmanet al. 2013). These and other approaches havealso been used to evaluate the importance ofrationally designed mutations.

Tumors from BRCA2 mutation carriers wereinitially reported to have lost the wild-typeBRCA2 allele in most cases (Collins et al. 1995;




http://www.research.nhgri.nih.gov/bic/





Gudmundsson et al. 1995). However, these find-ings were recently questioned by a study showingthat BRCA1 or BRCA2 loss of heterozygosity(LOH) does not always occur in breast tumors,and, when it does occur, LOH can involve themutant rather than the wild-type BRCA allele(King et al. 2007). In the mouse, germlineBrca2 heterozygous mutation promotes tumorformation in a Kras-driven mouse pancreaticcancer model, and loss of wild-type Brca2 alleleis not observed in some tumors (Skoulidis et al.2010). These studies open the possibility of hap-loinsufficiency of BRCA genes, although the dif-ferent approaches used in these studies and epi-genetic silencing of the remaining wild-typeallele could potentially account for some of theobservations.

Patients with biallelic BRCA2 mutationsare classified as having a Fanconi anemia sub-type (FA-D1), which is associated with brain,kidney, and hematological tumor types veryearly in life as well as with developmental issues(Howlett et al. 2002; Meyer et al. 2014). In thesecases, at least one of the BRCA2 alleles is expect-ed to be hypomorphic, given the requirement ofBrca2 during embryogenesis in mice (Moyna-han 2002).

BRCA2 Domains and Interactions

Human BRCA2 is a 3418 amino acid protein,which consists of multiple domains (Fig. 3). Inaddition to binding PALB2 (discussed below),BRCA2 binds RAD51 at motifs repeated in themiddle of the protein (BRC repeats) and at adistinct domain in the carboxyl terminus(Sharan et al. 1997; Wong et al. 1997). In addi-tion, BRCA2 binds DNA and the DSS1 proteinat a conserved region following the BRC repeats(Yang et al. 2002).

RAD51: Binding at the BRC Repeats andCarboxyl Terminus

Mammalian BRCA2 has eight BRC repeats,which are conserved among vertebrates inboth sequence and spacing, but not in the in-tervening sequences (Bignell et al. 1997). BRCrepeats appear to regulate RAD51 filament for-

mation in a complex manner. An individualBRC peptide, when present in excess, disruptsRAD51 filament formation in vitro (Davieset al. 2001) and interferes with damage-inducedRAD51 foci formation and HR in vivo (Chenet al. 1999; Xia et al. 2001; Stark et al. 2002; Saekiet al. 2006), presumably by mimicking the in-terface between RAD51 monomers within theRAD51 filament (Pellegrini et al. 2002). In con-trast, the full-length BRCA2 protein or the pep-tide containing all eight BRC repeats promotesRAD51-mediated strand exchange by stimu-lating assembly of RAD51 onto ssDNA, whilepreventing nucleation of RAD51 on dsDNA(Carreira et al. 2009; Jensen et al. 2010; Liuet al. 2010; Thorslund et al. 2010). Even a singleBRC4 motif has been shown to exhibit these ac-tivities under the appropriate experimental con-ditions (Carreira et al. 2009). The underlyingmechanism is thought to be through blockingRAD51-mediated ATP hydrolysis and, thus, sta-bilizing the active ATP-bound RAD51-ssDNAfilament. Evidence from Rad51 mutant chickencells indicates that Rad51 is sequestrated undernormal conditions by interaction with the BRCrepeats, but in response to damage, this seques-tered fraction undergoes mobilization, suggest-ing dynamic regulation of Rad51 through in-teraction with the BRC repeat region (Yu et al.2003a).

Genetic studies further established the im-portance of BRC repeats for BRCA2 function.Mice deleted for the BRC-encoding exon 11 inthe germline are inviable, whereas somatic exon11 deletion causes tumor development (Jonkerset al. 2001). Conversely, a mouse mutant thatmaintains the amino terminus of BRCA2 in-cluding just three BRC repeats can survive em-bryogenesis, albeit at low frequency (Friedmanet al. 1998). On the cellular level, the essentialityof BRC repeats for BRCA2 function is support-ed by proliferation defects observed in exon11-deleted embryonic fibroblasts (Bouwmanet al. 2010) and the failure to rescue defects inBRCA2-deficient cells with constructs devoid ofBRC repeats (Chen et al. 1998b). Point muta-tions in BRC repeats that impair RAD51 inter-action are found in breast cancer patients (Pel-legrini et al. 2002), although functional studies

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are required to determine the effects of thesemutations in full-length BRCA2.

Both functional redundancy and divergencemay exist among BRC repeats in the context offull-length BRCA2 protein. Redundancy amongBRC repeats is supported by the sufficiency ofan individual BRC repeat for HR function with-in the BRC-RPA fusion protein (see below;Saeki et al. 2006). A divergence of function issuggested by the poor sequence identity be-tween BRC repeats within a species, in contrastto high interspecies conservation of individualrepeats (Bignell et al. 1997). Consistent with thisnotion, electron microscopy shows that differ-ent BRC repeats bind to different regions ofRAD51 in filaments (Galkin et al. 2005). Bio-chemical studies led to the proposal of the ex-istence of two classes of BRC repeats: BRC1, 2, 3,and 4 bind to RAD51 monomers at high affin-ity and reduce the ATPase activity of RAD51,effectively targeting RAD51 onto ssDNA overdsDNA and stimulating RAD51-mediatedstrand exchange, whereas BRC5, 6, 7, and 8bind to the RAD51-ssDNA filament at high af-finity (Carreira and Kowalczykowski 2011).These results have led to a model whereby thefirst class of repeats facilitates nucleation ofRAD51 onto ssDNA and the second class stabi-lizes the nascent RAD51 nucleoprotein filament(Carreira and Kowalczykowski 2011).

The carboxy-terminal RAD51-binding siteof BRCA2 shares no homology with the BRCrepeats (Mizuta et al. 1997; Sharan et al.1997). RAD51 binding to this region is regulat-ed: it is abrogated by CDK phosphorylation atS3291 at G2/M, leading to the hypothesis thatit coordinates BRCA2 activity with cell-cycleprogression (Esashi et al. 2005). BRCA2 S3291is a key site for RAD51 binding, as both phos-phomimic (S3291E) and phosphodefective(S3291A) mutations block RAD51 interaction(Esashi et al. 2005). Unlike the BRC repeats, aBRCA2 carboxy-terminal peptide selectivelybinds RAD51 filaments at the interface regionbetween two RAD51 protomers, such thatcarboxy-terminal-binding functions to stabilizeRAD51-ssDNA filaments (Davies and Pellegrini2007; Esashi et al. 2007). Surprisingly, however,S3291 mutation confers little or no DNA-dam-

age sensitivity and does not compromise HR inthe context of full-length protein (Hucl et al.2008; Ayoub et al. 2009; Schlacher et al. 2011),although the mutation does compromise HRin crippled BRCA2 peptides that have defects inother functional domains (Siaud et al. 2011),implying that carboxy-terminal RAD51 bindingis not essential for HR but can promote HR un-der some circumstances. However, RAD51 bind-ing by the BRCA2 carboxyl terminus has beenimplicated in the protection of nascent DNAstrands at stalled replication forks (Schlacheret al. 2011).

DMC1, the meiosis-specific RAD51 homo-log, has been shown to have a distinct bindingsite on BRCA2 from the BRC repeats and car-boxyl terminus (a PheProPro motif ) (Thors-lund et al. 2007). However, disrupting the motifin the mouse does not have a discernible effecton DMC1 function and meiosis (Biswas et al.2012), suggesting that DMC1 binds elsewhere,perhaps at sites also bound by RAD51.

DNA and DSS1 Binding

That BRCA2 is a DNA-binding protein was re-vealed in structural studies (Yang et al. 2002).The DNA-binding domain (DBD) consists offive components: a helical domain, three oli-gonucleotide-binding (OB) folds that bindssDNA, and a tower domain with a three-helixbundle (3HB) at its end (Fig. 3). The 3HB issimilar to the DNA-binding domain of Hin re-combinase, suggesting dsDNA-binding activity.The helical domain, OB1 and OB2, interactswith the small, highly conserved DSS1 protein(Marston et al. 1999; Yang et al. 2002), whichhas been shown to promote HR in human cells(Gudmundsdottir et al. 2004; Li et al. 2006;Kristensen et al. 2010).

BRCA2 peptide mutants abrogated forDSS1 binding have impaired HR (Siaud et al.2011), indicating that at least part of the HRfunction of DSS1 is through interaction withBRCA2. Biochemical studies have shown thatDSS1 promotes the RAD51-loading activity ofBRCA2 (Liu et al. 2010). DSS1 also appears tomaintain the stability of BRCA2 protein in cells(Li et al. 2006), although an effect is not always





observed (Gudmundsdottir et al. 2004). In thefungus Ustilago maydis, the ortholog of DSS1 isalso required for HR by regulating Brh2, theU. maydis ortholog of BRCA2 (Kojic et al.2003, 2005). In budding yeast, which does nothave a BRCA2 homolog, the DSS1 homolog lo-calizes to DSB break sites and promotes HR-and NHEJ-mediated DSB repair, suggesting aBRCA2-independent function of DSS1 in DSBrepair (Krogan et al. 2004). DSS1 is also foundto interact with the 19S proteasome, the rele-vance of which to BRCA2 function remains tobe clarified.

Patient missense mutations are foundthroughout BRCA2 DBD domain (Yang et al.2002). Mutations predicted to compromise ei-ther the structural integrity of the DBD domainand/or DSS1/DNA-binding affect function(Kuznetsov et al. 2008; Biswas et al. 2011, 2012;Siaud et al. 2011). Moreover, mutation of ssDNAcontact residues or deletion of the 3HB has det-rimental effects on HR in reporter-based assaysmeasuring BRCA2 peptide activity (Siaud et al.2011). Surprisingly, a BRCA2 peptide deletedfor the entire DBD is still functional in HR(Siaud et al. 2011); in fact, deletion of the DBDis one type of reversion mutation identified forBRCA2 (Edwards et al. 2008). These findingsemphasize the plasticity of BRCA2 function inHR. An intact DBD is required if it is present, butloss of the entire DBD can be tolerated for sub-stantial HR function, as long as PALB2 interac-tion is intact (Siaud et al. 2011).

BRCA2 Function and HR

The earliest clues about the importance ofBRCA2 in maintaining genome integrity camefrom observations that Brca2 mutant miceshow early embryonic lethality and DNA repairdefects (Connor et al. 1997; Ludwig et al. 1997;Sharan et al. 1997; Suzuki et al. 1997) similar toRad51 mutant mice (Lim and Hasty 1996; Tsu-zuki et al. 1996). At the same time, BRCA2 in-teraction with RAD51 was uncovered (Sharanet al. 1997; Wong et al. 1997) and BRCA2 wasfound to colocalize with RAD51 in damage-induced nuclear foci (Chen et al. 1998a). Therequirement of BRCA2 in HR was directly dem-

onstrated using HR reporters (Fig. 2B) (Moy-nahan et al. 2001b).

BRCA2 clearly acts at a distinct step in HRfrom that of BRCA1 (Stark et al. 2004). Al-though BRCA1-BARD1 promotes repair byboth HR and SSA, BRCA2 promotes HR whilesuppressing SSA (Fig. 4) (Tutt et al. 2001; Starket al. 2004). End resection presumably initiatesnormally in BRCA2 mutant cells, but ssDNAoverhangs cannot be channeled into HR; in-stead, where present, complementary ssDNAoverhangs anneal to each other. RAD51 disrup-tion results in the same phenotype as BRCA2loss, indicating that genetically BRCA2 acts atthe same step in HR as RAD51 (Stark et al.2004). Not surprisingly then, loss of 53BP1does not rescue the viability of BRCA2-deficientcells, as it does with BRCA1-deficient cells(Bouwman et al. 2010).

Genetic and biochemical studies have alsopointed to a RAD51 mediator activity ofBRCA2. Remarkably, HR activity and genomicintegrity are restored to BRCA2-deficient ham-ster cells by fusing a single BRC motif to thessDNA-binding protein RPA (Saeki et al.2006), suggesting that the main role of BRCA2is to load RAD51 onto ssDNA. Purified Brh2protein, the BRCA2 ortholog in the fungus U.maydis, stimulates Rad51-mediated strand ex-change at an ssDNA-dsDNA junction by load-ing Rad51 onto DNA and displacing RPA (Yanget al. 2005). Recently, this RAD51-mediatorfunction was further confirmed with purifiedhuman BRCA2, which specifically promotesRAD51 filament assembly on ssDNA overdsDNA (Jensen et al. 2010; Liu et al. 2010; Thors-lund et al. 2010), leading to displacement of RPAto stimulate strand exchange (Jensen et al. 2010;Liu et al. 2010). Stabilization of RAD51-ssDNAcomplexes by BRCA2 occurs by inhibition of theDNA-dependent ATPase activity of RAD51. Al-though challenges exist studying this very largeprotein involved in multiple protein interac-tions, these studies are promising for under-standing the biochemistry of BRCA2.

Consistent with its major role in HR,BRCA2-deficient cells are sensitive to DNA-damaging agents that lead to lesions normallyrepaired by HR, including cross-linking agents

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(Kraakman-van der Zwet et al. 2002) and toPARP inhibitors (Bryant et al. 2005; Farmeret al. 2005). BRCA2 has also been implicatedin processes other than HR repair per se. Earlierstudies suggested that stalled replication forksare stabilized by BRCA2 (Lomonosov et al.2003). Recent work has provided evidence thatthis function is achieved through a RAD51-de-pendent, but HR-repair-independent mecha-nism, such that BRCA2 protects nascent DNAstrand from degradation by stabilizing theRAD51 filament to maintain genomic integrityunder replication stress (Schlacher et al. 2011).Roles for BRCA1 and canonical Fanconi anemiaproteins have also been shown in nascent strandprotection (Schlacher et al. 2012). The link be-tween BRCA1, BRCA2, and Fanconi anemiaproteins in replication fork protection is nota-ble because loss of canonical Fanconi anemiaproteins results in only mild HR defects inmammalian cells (Nakanishi et al. 2005), exceptfor HR repair of cross links coupled to replica-tion (Nakanishi et al. 2011).

PALB2

PALB2 Mutations in Patients

As with BRCA2, monoallelic PALB2 mutationsare associated with breast cancer susceptibility(Erkko et al. 2007; Rahman et al. 2007; Tisch-kowitz et al. 2007). The breast cancer risk asso-ciated with PALB2 mutation has recently beenestimated to overlap with that of BRCA2 muta-tion (Antoniou et al. 2014). Although muta-tions are generally infrequent compared withBRCA1 and BRCA2, in the Finnish population�1% of unselected breast cancers are associatedwith a founder PALB2 mutation (Erkko et al.2007). The clinical phenotype of breast cancerswith PALB2 mutation is more similar to thatof BRCA2 in that a more substantial fractionis estrogen receptor positive rather than triplenegative (Antoniou et al. 2014). MonoallelicPALB2 mutations have also been associatedwith pancreatic and ovarian cancer susceptibil-ity (Jones et al. 2009; Walsh et al. 2011).

Biallelic PALB2 mutation leads to a Fanconianemia subtype (FA-N), which shares a similar

tumor spectrum with the FA-D1 subtype aris-ing from BRCA2 mutation. Patients are predis-posed to developing early childhood cancers,such as Wilms’ tumor and medulloblastoma(Reid et al. 2007; Xia et al. 2007). Further, ho-mozygous germline deletion of Palb2 in miceleads to early embryonic lethality (Bouwmanet al. 2011; Bowman-Colin et al. 2013), whereasconditional deletion of Palb2 causes mammarytumors with long latency accelerated by p53 loss(Bowman-Colin et al. 2013; Huo et al. 2013). Aswith BRCA2 but unlike BRCA1, 53BP1 loss failsto rescue genome instability caused by PALB2deficiency (Bowman-Colin et al. 2013). Thesephenotypes are consistent with the notion thatPALB2 and BRCA2 function in the same step ofthe HR pathway to maintain genome integrityand suppress tumor development.

PALB2 Domains and Interactions

PALB2 is an 1186 amino acid protein with acoiled-coil domain at its amino terminus,which interacts with BRCA1, and a WD40 b-propeller domain at its carboxyl terminus,which interacts with BRCA2 (Fig. 3) (Xia et al.2006; Oliver et al. 2009; Sy et al. 2009; Zhanget al. 2009a,b). Emphasizing the importance ofthe PALB2 interaction for BRCA2 function, dis-ruption of this interaction results in severe HRdefects (Xia et al. 2006; Siaud et al. 2011). Fur-thermore, human BRCA2 mutations that abro-gate PALB2 interaction fail to support viabilityof Brca2-null mouse embryonic stem cells (Bis-was et al. 2012), although paradoxically Palb2-null embryonic stem cells have been reported tobe viable (Bowman-Colin et al. 2013).

PALB2 and HR

Biochemical studies have shown that purifiedPALB2 binds DNA and RAD51, and is able tostimulate RAD51-dependent strand invasion,including synergistically with a BRCA2 peptide(Buisson et al. 2010; Dray et al. 2010). A recentreport also links both BRCA2 and PALB2 to adownstream step in HR, DNA synthesis, bystimulating polymerase h activity on strand in-vasion intermediates (Buisson et al. 2014).





RAD51 PARALOGS

RAD51 Paralog Mutations in Patients

RAD51 has five paralogs (RAD51B, RAD51C,RAD51D, XRCC2, and XRCC3) that were dis-covered both as RAD51-related genes andthrough their complementation of radiation-sensitive Chinese hamster cell mutants (Tebbset al. 1995; Albala et al. 1997; Cartwright et al.1998; Dosanjh et al. 1998; Pittman et al. 1998).Thus far, the strongest evidence that RAD51paralogs are tumor suppressors comes fromstudies of RAD51C and RAD51D. Monoallelic,germline mutations in these genes predisposeto ovarian cancer (Meindl et al. 2010; Lovedayet al. 2011, 2012), although the predispositionto breast cancer is less clear. However, in bothcases, mutations have been observed in �1% offamilies with BRCA1/2-negative breast andovarian cancer. Truncating mutations havebeen reported for both RAD51C and RAD51Dand, in addition, missense mutations that im-pair the ATP-binding site have been reported forRAD51C (Meindl et al. 2010; Loveday et al.2011, 2012; Somyajit et al. 2012).

RAD51B, XRCC2, and XRCC3 monoallelicmutations have also been observed in breastcancer families, although the significance isnot certain (Hilbers et al. 2012; Park et al.2012; Golmard et al. 2013). One unusual find-ing is the presence of chromosomal transloca-tions involving RAD51B in some benign tumors(Ingraham et al. 1999; Schoenmakers et al.1999), including a germline translocation in afamily with multiple cases of thymoma (Nico-deme et al. 2005). As reduced gene dosage ofRAD51B has been reported to have phenotypicconsequences (Date et al. 2006), translocationsinvolving one allele may reduce repair activity.

Biallelic missense mutations of RAD51C(R258H) were identified in one consanguineousfamily with three children with severe congenitalabnormalities whose cells were sensitive to cross-linking agents (Vaz et al. 2010). Thus, biallelicRAD51C mutation is now considered to confera Fanconi anemia-like disorder, subtype FA-O.Two of the childrenwith RAD51C mutation diedas infants from the congenital defects; the sur-viving child was reported at 10 years of age to be

cancer free. Some of the congenital abnormali-ties are similar to those described in FA-D1 andFA-N children with biallelic BRCA2 and PALB2mutation, respectively; however, the absence of amalignancy in the olderchild is distinct (Moldo-van and D’Andrea 2009). RAD51C R258 is closeto the ATP-binding site and is highly conserved;the mutation has been shown to reduce butnot abolish HR function (Somyajit et al. 2012).XRCC2 is the only other RAD51 paralog besidesRAD51C for which a Fanconi anemia–like phe-notype has been described to date. A child froma consanguineous family with an XRCC2 trun-cating mutation has been reported with de-velopmental issues and cellular sensitivity tocross-linking agents (Shamseldin et al. 2012).

RAD51 Paralog Complexes

RAD51 paralogs show 20%–30% amino acidsequence similarity to RAD51 and with eachother, and this sequence conservation is pre-dominantly found at a globular domain withWalker A and B motifs, which are critical forthe ATP-binding/hydrolysis activity. Protein ho-mology modeling studies have predicted that theRAD51 paralogs, with the exception XRCC2,have an amino-terminal domain containinga four-helix bundle linked to the carboxy-ter-minal globular domain (Miller et al. 2004), sim-ilar to RAD51 (Shin et al. 2003). Yeast two-/three-hybrid, coimmunoprecipitation studies,and biochemical studies have shown thatRAD51 paralogs exist in two major complexes,RAD51B-RAD51C-RAD51D-XRCC2 (BCDX2)and RAD51C-XRCC3 (CX3) (Fig. 3), althoughsubcomplexes have also been identified (BC,CD, DX2, BCD, and CDX2) (Braybrooke et al.2000; Schild et al. 2000; Masson et al. 2001;Sigurdsson et al. 2001; Liu et al. 2002; Wieseet al. 2002). The amino-terminal domain ofone paralog apparently makes contact with thecarboxy-terminal domain of another, providingspecificity to the interactions (e.g., RAD51B-N-ter with RAD51C-C-ter, RAD51C-N-terwith RAD51D-C-ter, RAD51D-N-ter withXRCC2-C-ter) (Kurumizaka et al. 2003; Milleret al. 2004). Furthermore, RAD51 interactionswith the BCDX2 and CX3 complexes and indi-

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vidual paralogs have also been reported (Do-sanjh et al. 1998; Schild et al. 2000; Masson etal. 2001; Liu et al. 2002).

Biochemical activities of these various com-plexes and subcomplexes have been described,although their integration into HR pathwaysinvolving BRCA2 and other factors is notwell understood. The RAD51 paralogs bind avariety of DNA structures, including ssDNAand branched structures like Holliday junctions(Braybrooke et al. 2000; Kurumizaka et al. 2001;Masson et al. 2001; Sigurdsson et al. 2001; Yo-koyama et al. 2003, 2004; Liu et al. 2004). Asexpected, the RAD51 paralogs have been shownto hydrolyze ATP (Braybrooke et al. 2000; Si-gurdsson et al. 2001; Lio et al. 2003; Yokoyamaet al. 2003; Shim et al. 2004); an intact ATP hy-drolysis domain is required for the functionof several paralogs (French et al. 2003; Yamadaet al. 2004; Gruver et al. 2005; Wiese et al. 2006),although not for XRCC2 (O’Regan et al. 2001).The BC subcomplex has been reported to stim-ulate RAD51 strand-exchange activity in thepresence of RPA (Sigurdsson et al. 2001), appar-ently by stabilizing the RAD51 nucleoproteinfilament rather than facilitating RAD51 nucle-ation (Amunugama et al. 2013).

RAD51 Paralog Cellular Phenotypes

A key role for the RAD51 paralogs in HR wasinitially shown for XRCC2 and XRCC3 in Chi-nese hamster cell mutants (Johnson et al. 1999;Pierce et al. 1999) and later for each of the pa-ralogs in chicken B lymphocytes (Takata et al.2001). As with biochemical studies in humancells, two distinct paralog complexes are pre-dicted in chicken cells, given that mutationsin two paralogs in the same complex are epistat-ic (Rad51B and Rad51D), whereas mutationsin paralogs in two different complexes are not(Xrcc3 and Rad51D) (Yonetani et al. 2005).Overexpression of RAD51 in each of the chickencell mutants partially suppresses DNA-damagesensitivity, indicating a role for each of the pa-ralogs in RAD51 loading or filament stabiliza-tion (Takata et al. 2001). Overall, mammalianRAD51 paralog mutants display reduced DNA-damage-induced RAD51 focus formation and

have increased spontaneous chromosomal ab-normalities and are sensitive to DNA-damagingagents, such as cross-linking agents like cisplat-in, and to PARP inhibitors (Bishop et al. 1998;Liu et al. 1998; Cui et al. 1999; Takata et al. 2001;French et al. 2002; Yoshihara et al. 2004; Smi-raldo et al. 2005; Loveday et al. 2011; Urbin etal. 2012; Min et al. 2013).

An early role for RAD51 paralogs in HRpathways is supported by the rapid recruitmentof XRCC3 to DSBs, which is independent ofRAD51 (Forget et al. 2004). A later role in HRfor RAD51 paralogs has also been investigated.RAD51C accumulates in nuclear foci in re-sponse to ionizing radiation where it colocalizeswith RAD51; however, RAD51C foci persistfor longer than RAD51 foci, suggesting thatRAD51C, a component of both main complexes(BCDX2 and CX3), has a role in both earlyand late stages of HR (Badie et al. 2009). Likehamster and chicken cells, XRCC3 knockoutHCT116 human cells were initially reported tohave impaired RAD51 focus formation (Yoshi-hara et al. 2004); however, a recent study in thesecells and in XRCC3 siRNA knockdown MCF7cells reported no RAD51 focus formation de-fect, leading investigators to conclude thatBCDX2 acts to load RAD51 onto DNA endswhile CX3 acts after RAD51 loading (Chunet al. 2013). Consistent with a distinct role forXRCC3 among the paralogs, XRCC3 knock-down in HeLa cells results in a persistent spin-dle assembly checkpoint, whereas RAD51B orRAD51C knockdown induces a G2/M cell-cy-cle arrest (Rodrigue et al. 2013). Overall, thesestudies point to the need for a coherent mam-malian model to dissect the roles of the individ-ual paralogs in HR.

RAD51 Paralog Mouse Knockout Models

Mouse knockouts of four RAD51 paralogs(Rad51b, Rad51c, Rad51d, and Xrcc2) havebeen reported and each is embryonic lethal,implying a critical function at developmentalstages when cells are rapidly dividing (Shuet al. 1999; Deans et al. 2000; Pittman and Schi-menti 2000; Kuznetsov et al. 2009; Smeenk et al.2010). No mouse model for Xrcc3 is currently





available. The reported developmental stage ofdeath is not equivalent in the various knock-outs, suggesting possible distinct functions ofthe paralogs, although differences in geneticbackground of the various mutants may ac-count for some of the phenotypic differences.Embryonic lethality does not appear to be de-fined by a specific developmental defect but,rather, the embryos show growth delays overthe course of a few days, consistent with whatmight be expected from stochastic DNA dam-age that is not repaired.

Supporting repair defects leading to apo-ptosis as a cause of lethality, death is delayed afew days by Trp53 mutation, although postnatalviability is not restored (Shu et al. 1999; Smir-aldo et al. 2005; Adam et al. 2007; Kuznetsovet al. 2009). The partial rescue by Trp53 muta-tion is similar to that observed with Rad51,Brca1, and Brca2 mutants (Moynahan 2002)but is unlike NHEJ mutants, in which the lateembryonic lethality is rescued to give rise toviable (although feeble) mice (Frank et al.2000; Gao et al. 2000). The one exception isXrcc2, in which the viability of one of the tworeported knockouts is rescued by Trp53 muta-tion (see below) (Orii et al. 2006).

A summary of the RAD51 paralog knock-outs is as follows in order of embryonic death(Rad51b, Rad51c, Rad51d, Xrcc2):

Rad51b mutants have severe growth retardationby embryonic day E5.5 and by E8.5 arecompletely resorbed (Shu et al. 1999). Em-bryonic stem cell lines could not be derived,suggesting that loss of RAD51B is cell lethal,similar to RAD51, BRCA1, or BRCA2 (Moy-nahan 2002).

Rad51c mutants show abnormalities by E5.5,are severely retarded by E8.5, and are re-sorbed shortly thereafter (Kuznetsov et al.2009). Rad51c and Trp53 are on the samechromosome, and adult mice that are hetero-zygous for both genes in cis develop a tumorspectrum (including mammary and prepu-tial gland tumors) that differs from mice ei-ther with both mutations in trans or withTrp53 mutation alone. The difference in thecis and trans mice is because LOH allows tu-

mors to develop, which are null for bothRad51c and Trp53. A hypomorphic Rad51cmutant has also been described in whichRAD51C levels are reduced significantly(�30%) (Kuznetsov et al. 2007). This levelof expression is sufficient to give rise to viablemice; however, a significant fraction is infer-tile owing to meiotic defects, including re-duced RAD51 foci and the presence of unre-paired DSBs in spermatocytes.

Rad51d embryos display a range of abnormali-ties between E7.5 and 10.5, and none areviable by E11.5 (Pittman and Schimenti2000). Trp53 mutation leads to a significantrescue, such that embryos are detected as lateas E16.5, although they are growth retarded(Smiraldo et al. 2005). Rad51d is located be-tween Rad51c and Trp53; mice with Rad51dand Trp53 heterozygous mutations in cis arenot tumor prone up to 12 months of age.

Xrcc2 mutants develop normally to E8.5; there-after, they display developmental delay/de-fects and are found at sub-Mendelian ratios(Deans et al. 2000). The few mice that areborn succumb soon after birth. However,when Xrcc2 mice are backcrossed onto aC57BL/6 genetic background, embryos donot reach late embryogenesis, although com-bined Trp53 mutation allows some embryosto survive until just before birth (Adam et al.2007). In another knockout model, presum-ably on a mixed genetic background, Trp53mutation actually rescues the viability ofXrcc2 mice (Orii et al. 2006).

Apoptosis is elevated throughout Xrcc2 mu-tant embryos (Deans et al. 2000), especially inthe brain in proliferating cells, which is distinctfrom Lig4 mutant embryos where it predomi-nates in postmitotic cells (Fig. 2C) (Orii et al.2006). Trp53 mutation suppresses apoptosis inthe developing brain and rescues the viability ofXrcc2 mutant mice in this model, as it does toLig4 mutant mice (Frank et al. 2000; Orii et al.2006), permitting a comparison of postnatalphenotypes, such as tumorigenesis. AlthoughLig4/Trp53 null mice succumb to Myc-ampli-fied B cell lymphoma and medulloblastoma by

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3 months of age (Frank et al. 2000; Lee andMcKinnon 2002), Xrcc2/Trp53 null mice havea broad tumor spectrum with an earlier onsetwithout obvious Myc amplification (Orii et al.2006). Thus, both HR and NHEJ are importantfor the viability of mice and tumor suppression,although their roles differ.

Rad51c/Trp53, Rad51d/Trp53, and Xrcc2/Trp53 mouse embryonic fibroblasts have beenestablished (Smiraldo et al. 2005; Adam et al.2007; Kuznetsov et al. 2009), as have Xrcc2 em-bryonic stem cell lines (Deans et al. 2003). TheseRAD51 paralog mutant cells are sensitive toDNA-damaging agents, such as ionizing radia-tion and interstrand cross-links, have defects inRAD51 foci formation, increased genomic in-stability, and, where checked, decreased HR inreporter assays. Rad51d/Trp53 fibroblasts haveshortened telomeres, suggesting that RAD51Dplays a role in telomere maintenance (Tarsounaset al. 2004).

SUMMARY AND FUTURE DIRECTIONS

Genetic studies have revealed a link betweengermline mutations in several HR genes andpredisposition to breast/ovarian and other tu-mors. Somatic mutations in HR genes have alsobeen uncovered (Cancer Genome Atlas Re-search Network 2011; Pennington et al. 2014).Insight into the molecular functions of the var-ious HR proteins has been forthcoming. A rolefor BRCA1 in the end resection step of HR iswell supported. At least three mechanisms areimplicated: antagonizing the resection inhibitor53BP1, promoting resection in the BRCA1-Ccomplex with CtIP, and inhibiting resectionin the BRCA1-A complex through Abraxas-RAP80. The BRCA1-PALB2-BRCA2 complexand RAD51 paralogs cooperate to load RAD51onto ssDNA coated with RPA to form the essen-tial recombination intermediate, the RAD51-ssDNA filament. The crucial role of these genesin key steps of HR provides important clues forunderstanding the cause of cancer in patientswith mutations in these genes and for investi-gating more effective cancer treatments.

However, many key questions remain to beaddressed. For instance, how are the various

roles of BRCA1 orchestrated? How do so manyplayers, including the BRCA1-PALB2-BRCA2complex and RAD51 paralog complexes, collab-orate to load and stabilize RAD51 onto ssDNA?Importantly, how exactly does HR deficiencyspecifically contribute to ovarian/breast carci-nogenesis and other cancer-prone diseases,such as Fanconi anemia? And how are otherpotential functions of HR proteins integratedinto their tumor suppressor roles? A compre-hensive understanding of disease-linked com-ponents and mechanisms of HR in mammaliansystem will be vital, including for therapeuticapproaches that target the HR pathway.

ACKNOWLEDGMENTS

We thank previous members of the Jasin Labo-ratory whose work is referenced in this review.This work is supported by National Institutes ofHealth GM110978 (to R.P.) and CA185660 andGM054668 (to M.J.).

REFERENCES�Reference is also in this collection.

Adam J, Deans B, Thacker J. 2007. A role for Xrcc2 in theearly stages of mouse development. DNA Repair (Amst)6: 224–234.

Albala JS, Thelen MP, Prange C, Fan W, Christensen M,Thompson LH, Lennon GG. 1997. Identification of anovel human RAD51 homolog, RAD51B. Genomics 46:476–479.

Amunugama R, Groden J, Fishel R. 2013. The HsRAD51B-HsRAD51C stabilizes the HsRAD51 nucleoprotein fila-ment. DNA Repair (Amst) 12: 723–732.

Antoniou A, Pharoah PD, Narod S, Risch HA, Eyfjord JE,Hopper JL, Loman N, Olsson H, Johannsson O, Borg A,et al. 2003. Average risks of breast and ovarian cancerassociated with BRCA1 or BRCA2 mutations detectedin case series unselected for family history: A combinedanalysis of 22 studies. Am J Hum Genet 72: 1117–1130.

Antoniou AC, Casadei S, Heikkinen T, Barrowdale D, PylkasK, Roberts J, Lee A, Subramanian D, De Leeneer K, Fos-tira F, et al. 2014. Breast-cancer risk in families with mu-tations in PALB2. N Engl J Med 371: 497–506.

Ayoub N, Rajendra E, Su X, Jeyasekharan AD, Mahen R,Venkitaraman AR. 2009. The carboxyl terminus ofBrca2 links the disassembly of Rad51 complexes to mi-totic entry. Curr Biol 19: 1075–1085.

Badie S, Liao C, Thanasoula M, Barber P, Hill MA, Tarsou-nas M. 2009. RAD51C facilitates checkpoint signaling bypromoting CHK2 phosphorylation. J Cell Biol 185: 587–600.





Bignell G, Micklem G, Stratton MR, Ashworth A, WoosterR. 1997. The BRC repeats are conserved in mammalianBRCA2 proteins. Hum Mol Genet 6: 53–58.

Bishop DK, Ear U, Bhattacharyya A, Calderone C, BeckettM, Weichselbaum RR, Shinohara A. 1998. Xrcc3 is re-quired for assembly of Rad51 complexes in vivo. J BiolChem 273: 21482–21488.

Biswas K, Das R, Alter BP, Kuznetsov SG, Stauffer S, NorthSL, Burkett S, Brody LC, Meyer S, Byrd RA, et al. 2011. Acomprehensive functional characterization of BRCA2variants associated with Fanconi anemia using mouseES cell-based assay. Blood 118: 2430–2442.

Biswas K, Das R, Eggington JM, Qiao H, North SL, StaufferS, Burkett SS, Martin BK, Southon E, Sizemore SC, et al.2012. Functional evaluation of BRCA2 variants mappingto the PALB2-binding and C-terminal DNA-binding do-mains using a mouse ES cell-based assay. Hum Mol Genet21: 3993–4006.

� Bizard AH, Hickson ID. 2014. The dissolution of doubleHolliday junctions. Cold Spring Harb Perspect Biol 6:a016477.

Bouwman P, Aly A, Escandell JM, Pieterse M, Bartkova J,van der Gulden H, Hiddingh S, Thanasoula M, KulkarniA, Yang Q, et al. 2010. 53BP1 loss rescues BRCA1 defi-ciency and is associated with triple-negative and BRCA-mutated breast cancers. Nat Struct Mol Biol 17: 688–695.

Bouwman P, Drost R, Klijn C, Pieterse M, van der Gulden H,Song JY, Szuhai K, Jonkers J. 2011. Loss of p53 partiallyrescues embryonic development of Palb2 knockout micebut does not foster haploinsufficiency of Palb2 in tumoursuppression. J Pathol 224: 10–21.

Bouwman P, van der Gulden H, van der Heijden I, Drost R,Klijn CN, Prasetyanti P, Pieterse M, Wientjens E, Seibler J,Hogervorst FB, et al. 2013. A high-throughput functionalcomplementation assay for classification of BRCA1 mis-sense variants. Cancer Discov 3: 1142–1155.

Bowman-Colin C, Xia B, Bunting S, Klijn C, Drost R, Bouw-man P, Fineman L, Chen X, Culhane AC, Cai H, et al.2013. Palb2 synergizes with Trp53 to suppress mammarytumor formation in a model of inherited breast cancer.Proc Natl Acad Sci 110: 8632–8637.

Braybrooke JP, Spink KG, Thacker J, Hickson ID. 2000. TheRAD51 family member, RAD51L3, is a DNA-stimulatedATPase that forms a complex with XRCC2. J Biol Chem275: 29100–29106.

Bryant HE, Schultz N, Thomas HD, Parker KM, Flower D,Lopez E, Kyle S, Meuth M, Curtin NJ, Helleday T. 2005.Specific killing of BRCA2-deficient tumours with inhib-itors of poly(ADP-ribose) polymerase. Nature 434: 913–917.

Brzovic PS, Rajagopal P, Hoyt DW, King MC, Klevit RE.2001. Structure of a BRCA1-BARD1 heterodimericRING–RING complex. Nat Struct Biol 8: 833–837.

Buis J, Stoneham T, Spehalski E, Ferguson DO. 2012. Mre11regulates CtIP-dependent double-strand break repair byinteraction with CDK2. Nat Struct Mol Biol 19: 246–252.

Buisson R, Dion-Cote AM, Coulombe Y, Launay H, Cai H,Stasiak AZ, Stasiak A, Xia B, Masson JY. 2010. Coopera-tion of breast cancer proteins PALB2 and piccolo BRCA2in stimulating homologous recombination. Nat StructMol Biol 17: 1247–1254.

Buisson R, Niraj J, Pauty J, Maity R, Zhao W, Coulombe Y,Sung P, Masson JY. 2014. Breast cancer proteins PALB2and BRCA2 stimulate polymerase h in recombination-associated DNA synthesis at blocked replication forks.Cell Rep 6: 553–564.

Bunting SF, Callen E, Wong N, Chen HT, Polato F, Gunn A,Bothmer A, Feldhahn N, Fernandez-Capetillo O, Cao L,et al. 2010. 53BP1 inhibits homologous recombinationin Brca1-deficient cells by blocking resection of DNAbreaks. Cell 141: 243–254.

Bunting SF, Callen E, Kozak ML, Kim JM, Wong N, Lopez-Contreras AJ, Ludwig T, Baer R, Faryabi RB, MalhowskiA, et al. 2012. BRCA1 functions independently of homol-ogous recombination in DNA interstrand crosslink re-pair. Mol Cell 46: 125–135.

Caleca L, Putignano AL, Colombo M, Congregati C, SarkarM, Magliery TJ, Ripamonti CB, Foglia C, Peissel B, Zaf-faroni D, et al. 2014. Characterization of an Italian foun-der mutation in the RING-finger domain of BRCA1.PLoS ONE 9: e86924.

Cancer Genome Atlas Research Network. 2011. Integratedgenomic analyses of ovarian carcinoma. Nature 474:609–615.

Cantor SB, Bell DW, Ganesan S, Kass EM, Drapkin R, Gross-man S, Wahrer DC, Sgroi DC, Lane WS, Haber DA, et al.2001. BACH1, a novel helicase-like protein, interacts di-rectly with BRCA1 and contributes to its DNA repairfunction. Cell 105: 149–160.

Cantor S, Drapkin R, Zhang F, Lin Y, Han J, Pamidi S,Livingston DM. 2004. The BRCA1-associated proteinBACH1 is a DNA helicase targeted by clinically relevantinactivating mutations. Proc Natl Acad Sci 101: 2357–2362.

Carreira A, Kowalczykowski SC. 2011. Two classes of BRCrepeats in BRCA2 promote RAD51 nucleoprotein fila-ment function by distinct mechanisms. Proc Natl AcadSci 108: 10448–10453.

Carreira A, Hilario J, Amitani I, Baskin RJ, Shivji MK, Ven-kitaraman AR, Kowalczykowski SC. 2009. The BRC re-peats of BRCA2 modulate the DNA-binding selectivity ofRAD51. Cell 136: 1032–1043.

Cartwright R, Tambini CE, Simpson PJ, Thacker J. 1998.The XRCC2 DNA repair gene from human and mouseencodes a novel member of the recA/RAD51 family. Nu-cleic Acids Res 26: 3084–3089.

Chang S, Biswas K, Martin BK, Stauffer S, Sharan SK. 2009.Expression of human BRCA1 variants in mouse ES cellsallows functional analysis of BRCA1 mutations. J ClinInvest 119: 3160–3171.

Chapman JR, Sossick AJ, Boulton SJ, Jackson SP. 2012a.BRCA1-associated exclusion of 53BP1 from DNA dam-age sites underlies temporal control of DNA repair. J CellSci 125: 3529–3534.

Chapman JR, Taylor MR, Boulton SJ. 2012b. Playing the endgame: DNA double-strand break repair pathway choice.Mol Cell 47: 497–510.

Chapman JR, Barral P, Vannier JB, Borel V, Steger M, Tomas-Loba A, Sartori AA, Adams IR, Batista FD, Boulton SJ.2013. RIF1 is essential for 53BP1-dependent nonhomol-ogous end joining and suppression of DNA double-strand break resection. Mol Cell 49: 858–871.

R. Prakash et al.




Chen J, Silver DP, Walpita D, Cantor SB, Gazdar AF, Tom-linson G, Couch FJ, Weber BL, Ashley T, Livingston DM,et al. 1998a. Stable interaction between the products ofthe BRCA1 and BRCA2 tumor suppressor genes in mi-totic and meiotic cells. Mol Cell 2: 317–328.

Chen PL, Chen CF, Chen Y, Xiao J, Sharp ZD, Lee WH.1998b. The BRC repeats in BRCA2 are critical forRAD51 binding and resistance to methyl methanesulfo-nate treatment. Proc Natl Acad Sci 95: 5287–5292.

Chen CF, Chen PL, Zhong Q, Sharp ZD, Lee WH. 1999.Expression of BRC repeats in breast cancer cells disruptsthe BRCA2-Rad51 complex and leads to radiation hyper-sensitivity and loss of G2/M checkpoint control. J BiolChem 274: 32931–32935.

Chen L, Nievera CJ, Lee AY, Wu X. 2008. Cell cycle-depen-dent complex formation of BRCA1.CtIP.MRN is impor-tant for DNA double-strand break repair. J Biol Chem283: 7713–7720.

Choi E, Park PG, Lee HO, Lee YK, Kang GH, Lee JW, Han W,Lee HC, Noh DY, Lekomtsev S, et al. 2012. BRCA2 fine-tunes the spindle assembly checkpoint through reinforce-ment of BubR1 acetylation. Dev Cell 22: 295–308.

Christensen DE, Brzovic PS, Klevit RE. 2007. E2-BRCA1RING interactions dictate synthesis of mono- or specificpolyubiquitin chain linkages. Nat Struct Mol Biol 14:941–948.

Chun J, Buechelmaier ES, Powell SN. 2013. Rad51 paralogcomplexes BCDX2 and CX3 act at different stages in theBRCA1-BRCA2-dependent homologous recombinationpathway. Mol Cell Biol 33: 387–395.

Coleman KA, Greenberg RA. 2011. The BRCA1-RAP80complex regulates DNA repair mechanism utilizationby restricting end resection. J Biol Chem 286: 13669–13680.

Collins N, McManus R, Wooster R, Mangion J, Seal S,Lakhani SR, Ormiston W, Daly PA, Ford D, Easton DF,et al. 1995. Consistent loss of the wild type allele in breastcancers from a family linked to the BRCA2 gene on chro-mosome 13q12–13. Oncogene 10: 1673–1675.

Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD,Wu X, Jiang W, Marraffini LA, et al. 2013. Multiplexgenome engineering using CRISPR/Cas systems. Science339: 819–823.

Connor F, Bertwistle D, Mee PJ, Ross GM, Swift S, Grigor-ieva E, Tybulewicz VL, Ashworth A. 1997. Tumorigenesisand a DNA repair defect in mice with a truncating Brca2mutation. Nat Genet 17: 423–430.

Consortium BCL. 1999. Cancer risks in BRCA2 mutationcarriers. J Natl Cancer Inst 91: 1310–1316.

Couedel C, Mills KD, Barchi M, Shen L, Olshen A, JohnsonRD, Nussenzweig A, Essers J, Kanaar R, Li GC, et al. 2004.Collaboration of homologous recombination and non-homologous end-joining factors for the survival and in-tegrity of mice and cells. Genes Dev 18: 1293–1304.

Cox MM, Goodman MF, Kreuzer KN, Sherratt DJ, SandlerSJ, Marians KJ. 2000. The importance of repairing stalledreplication forks. Nature 404: 37–41.

Cui X, Brenneman M, Meyne J, Oshimura M, Goodwin EH,Chen DJ. 1999. The XRCC2 and XRCC3 repair genes arerequired for chromosome stability in mammalian cells.Mutat Res 434: 75–88.

� Daley JM, Gaines WA, Kwon YH, Sung P. 2014. Regulation ofDNA pairing in homologous recombination. Cold SpringHarb Perspect Biol 6: a017954.

D’Andrea AD. 2013. BRCA1: A missing link in the Fanconianemia/BRCA pathway. Cancer Discov 3: 376–378.

Daniels MJ, Wang Y, Lee M, Venkitaraman AR. 2004. Ab-normal cytokinesis in cells deficient in the breast cancersusceptibility protein BRCA2. Science 306: 876–879.

Date O, Katsura M, Ishida M, Yoshihara T, Kinomura A,Sueda T, Miyagawa K. 2006. Haploinsufficiency ofRAD51B causes centrosome fragmentation and aneu-ploidy in human cells. Cancer Res 66: 6018–6024.

Daugaard M, Baude A, Fugger K, Povlsen LK, Beck H, Sor-ensen CS, Petersen NH, Sorensen PH, Lukas C, Bartek J,et al. 2012. LEDGF (p75) promotes DNA-end resectionand homologous recombination. Nat Struct Mol Biol 19:803–810.

Davies OR, Pellegrini L. 2007. Interaction with the BRCA2 Cterminus protects RAD51-DNA filaments from disas-sembly by BRC repeats. Nat Struct Mol Biol 14: 475–483.

Davies AA, Masson JY, McIlwraith MJ, Stasiak AZ, Stasiak A,Venkitaraman AR, West SC. 2001. Role of BRCA2 in con-trol of the RAD51 recombination and DNA repair pro-tein. Mol Cell 7: 273–282.

Deans B, Griffin CS, Maconochie M, Thacker J. 2000. Xrcc2is required for genetic stability, embryonic neurogenesisand viability in mice. EMBO J 19: 6675–6685.

Deans B, Griffin CS, O’Regan P, Jasin M, Thacker J. 2003.Homologous recombination deficiency leads to pro-found genetic instability in cells derived from Xrcc2-knockout mice. Cancer Res 63: 8181–8187.

De Brakeleer S, De Greve J, Loris R, Janin N, Lissens W,Sermijn E, Teugels E. 2010. Cancer predisposing missenseand protein truncating BARD1 mutations in non-BRCA1or BRCA2 breast cancer families. Hum Mutat 31: E1175–E1185.

Dever SM, Golding SE, Rosenberg E, Adams BR, Idowu MO,Quillin JM, Valerie N, Xu B, Povirk LF, Valerie K. 2011.Mutations in the BRCT binding site of BRCA1 result inhyper-recombination. Aging (Albany NY) 3: 515–532.

Doil C, Mailand N, Bekker-Jensen S, Menard P, Larsen DH,Pepperkok R, Ellenberg J, Panier S, Durocher D, Bartek J,et al. 2009. RNF168 binds and amplifies ubiquitin con-jugates on damaged chromosomes to allow accumulationof repair proteins. Cell 136: 435–446.

Domchek SM, Tang J, Stopfer J, Lilli DR, Hamel N, Tisch-kowitz M, Monteiro AN, Messick TE, Powers J, Yonker A,et al. 2013. Biallelic deleterious BRCA1 mutations in awoman with early-onset ovarian cancer. Cancer Discov 3:399–405.

Dosanjh MK, Collins DW, Fan W, Lennon GG, Albala JS,Shen Z, Schild D. 1998. Isolation and characterization ofRAD51C, a new human member of the RAD51 family ofrelated genes. Nucleic Acids Res 26: 1179–1184.

Dray E, Etchin J, Wiese C, Saro D, Williams GJ, Hammel M,Yu X, Galkin VE, Liu D, Tsai MS, et al. 2010. Enhance-ment of RAD51 recombinase activity by the tumor sup-pressor PALB2. Nat Struct Mol Biol 17: 1255–1259.

Drost R, Bouwman P, Rottenberg S, Boon U, Schut E, Klar-enbeek S, Klijn C, van der Heijden I, van der Gulden H,Wientjens E, et al. 2011. BRCA1 RING function is essen-





tial for tumor suppression but dispensable for therapyresistance. Cancer Cell 20: 797–809.

Edwards SM, Kote-Jarai Z, Meitz J, Hamoudi R, Hope Q,Osin P, Jackson R, Southgate C, Singh R, Falconer A, et al.2003. Two percent of men with early-onset prostate can-cer harbor germline mutations in the BRCA2 gene. Am JHum Genet 72: 1–12.

Edwards SL, Brough R, Lord CJ, Natrajan R, Vatcheva R,Levine DA, Boyd J, Reis-Filho JS, Ashworth A. 2008.Resistance to therapy caused by intragenic deletion inBRCA2. Nature 451: 1111–1115.

Erkko H, Xia B, Nikkila J, Schleutker J, Syrjakoski K, Man-nermaa A, Kallioniemi A, Pylkas K, Karppinen SM, Ra-pakko K, et al. 2007. A recurrent mutation in PALB2 inFinnish cancer families. Nature 446: 316–319.

Esashi F, Christ N, Gannon J, Liu Y, Hunt T, Jasin M, WestSC. 2005. CDK-dependent phosphorylation of BRCA2 asa regulatory mechanism for recombinational repair. Na-ture 434: 598–604.

Esashi F, Galkin VE, Yu X, Egelman EH, West SC. 2007.Stabilization of RAD51 nucleoprotein filaments by theC-terminal region of BRCA2. Nat Struct Mol Biol 14:468–474.

Escribano-Diaz C, Orthwein A, Fradet-Turcotte A, Xing M,Young JT, Tkac J, Cook MA, Rosebrock AP, Munro M,Canny MD, et al. 2013. A cell cycle-dependent regulatorycircuit composed of 53BP1-RIF1 and BRCA1-CtIP con-trols DNA repair pathway choice. Mol Cell 49: 872–883.

Essers J, Hendriks RW, Swagemakers SM, Troelstra C, de WitJ, Bootsma D, Hoeijmakers JH, Kanaar R. 1997. Disrup-tion of mouse RAD54 reduces ionizing radiation resis-tance and homologous recombination. Cell 89: 195–204.

Farmer H, McCabe N, Lord CJ, Tutt AN, Johnson DA,Richardson TB, Santarosa M, Dillon KJ, Hickson I,Knights C, et al. 2005. Targeting the DNA repair defectin BRCA mutant cells as a therapeutic strategy. Nature434: 917–921.

Feng L, Fong KW, Wang J, Wang W, Chen J. 2013. RIF1counteracts BRCA1-mediated end resection duringDNA repair. J Biol Chem 288: 11135–11143.

Forget AL, Bennett BT, Knight KL. 2004. Xrcc3 is recruitedto DNA double strand breaks early and independent ofRad51. J Cell Biochem 93: 429–436.

Foulkes WD, Stefansson IM, Chappuis PO, Begin LR, GoffinJR, Wong N, Trudel M, Akslen LA. 2003. GermlineBRCA1 mutations and a basal epithelial phenotype inbreast cancer. J Natl Cancer Inst 95: 1482–1485.

Frank KM, Sharpless NE, Gao Y, Sekiguchi JM, FergusonDO, Zhu C, Manis JP, Horner J, DePinho RA, Alt FW.2000. DNA ligase IV deficiency in mice leads to defectiveneurogenesis and embryonic lethality via the p53 path-way. Mol Cell 5: 993–1002.

French CA, Masson JY, Griffin CS, O’Regan P, West SC,Thacker J. 2002. Role of mammalian RAD51L2(RAD51C) in recombination and genetic stability. J BiolChem 277: 19322–19330.

French CA, Tambini CE, Thacker J. 2003. Identification offunctional domains in the RAD51L2 (RAD51C) proteinand its requirement for gene conversion. J Biol Chem 278:45445–45450.

Friedman LS, Thistlethwaite FC, Patel KJ, Yu VP, Lee H,Venkitaraman AR, Abel KJ, Carlton MB, Hunter SM,Colledge WH, et al. 1998. Thymic lymphomas in micewith a truncating mutation in Brca2. Cancer Res 58:1338–1343.

Futreal PA, Liu Q, Shattuck-Eidens D, Cochran C, Harsh-man K, Tavtigian S, Bennett LM, Haugen-Strano A,Swensen J, Miki Y, et al. 1994. BRCA1 mutations in pri-mary breast and ovarian carcinomas. Science 266: 120–122.

Galkin VE, Esashi F, Yu X, Yang S, West SC, Egelman EH.2005. BRCA2 BRC motifs bind RAD51-DNA filaments.Proc Natl Acad Sci 102: 8537–8542.

Gao Y, Ferguson DO, Xie W, Manis JP, Sekiguchi J, FrankKM, Chaudhuri J, Horner J, DePinho RA, Alt FW. 2000.Interplay of p53 and DNA-repair protein XRCC4 in tu-morigenesis, genomic stability and development. Nature404: 897–900.

Golmard L, Caux-Moncoutier V, Davy G, Al Ageeli E, PoirotB, Tirapo C, Michaux D, Barbaroux C, d’Enghien CD,Nicolas A, et al. 2013. Germline mutation in the RAD51Bgene confers predisposition to breast cancer. BMC Cancer13: 484.

Greenberg RA, Sobhian B, Pathania S, Cantor SB, NakataniY, Livingston DM. 2006. Multifactorial contributions toan acute DNA damage response by BRCA1/BARD1-con-taining complexes. Genes Dev 20: 34–46.

Gruver AM, Miller KA, Rajesh C, Smiraldo PG, Kaliyaper-umal S, Balder R, Stiles KM, Albala JS, Pittman DL. 2005.The ATPase motif in RAD51D is required for resistance toDNA interstrand crosslinking agents and interaction withRAD51C. Mutagenesis 20: 433–440.

Gudmundsdottir K, Lord CJ, Witt E, Tutt AN, Ashworth A.2004. DSS1 is required for RAD51 focus formation andgenomic stability in mammalian cells. EMBO Rep 5:989–993.

Gudmundsson J, Johannesdottir G, Bergthorsson JT, Ara-son A, Ingvarsson S, Egilsson V, Barkardottir RB. 1995.Different tumor types from BRCA2 carriers show wild-type chromosome deletions on 13q12-q13. Cancer Res55: 4830–4832.

Hilbers FS, Wijnen JT, Hoogerbrugge N, Oosterwijk JC,Collee MJ, Peterlongo P, Radice P, Manoukian S, FeroceI, Capra F, et al. 2012. Rare variants in XRCC2 as breastcancer susceptibility alleles. J Med Genet 49: 618–620.

Howlett NG, Taniguchi T, Olson S, Cox B, Waisfisz Q, DeDie-Smulders C, Persky N, Grompe M, Joenje H, Pals G,et al. 2002. Biallelic inactivation of BRCA2 in Fanconianemia. Science 297: 606–609.

Hu Y, Scully R, Sobhian B, Xie A, Shestakova E, LivingstonDM. 2011. RAP80-directed tuning of BRCA1 homolo-gous recombination function at ionizing radiation-in-duced nuclear foci. Genes Dev 25: 685–700.

Hucl T, Rago C, Gallmeier E, Brody JR, Gorospe M, Kern SE.2008. A syngeneic variance library for functional anno-tation of human variation: Application to BRCA2. CancerRes 68: 5023–5030.

Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB, ChenJ. 2007. RNF8 transduces the DNA-damage signal viahistone ubiquitylation and checkpoint protein assembly.Cell 131: 901–914.

R. Prakash et al.




Huen MS, Sy SM, Chen J. 2010. BRCA1 and its toolbox forthe maintenance of genome integrity. Nat Rev Mol CellBiol 11: 138–148.

Huo Y, Cai H, Teplova I, Bowman-Colin C, Chen G, Price S,Barnard N, Ganesan S, Karantza V, White E, et al. 2013.Autophagy opposes p53-mediated tumor barrier to fa-cilitate tumorigenesis in a model of PALB2-associatedhereditary breast cancer. Cancer Discov 3: 894–907.

Ingraham SE, Lynch RA, Kathiresan S, Buckler AJ, MenonAG. 1999. hREC2, a RAD51-like gene, is disrupted byt(12;14) (q15;q24.1) in a uterine leiomyoma. Cancer Ge-net Cytogenet 115: 56–61.

Jasin M, Rothstein R. 2013. Repair of strand breaks by ho-mologous recombination. Cold Spring Harb Perspect Biol5: a012740.

Jensen RB, Carreira A, Kowalczykowski SC. 2010. Purifiedhuman BRCA2 stimulates RAD51-mediated recombina-tion. Nature 467: 678–683.

Johnson RD, Jasin M. 2000. Sister chromatid gene conver-sion is a prominent double-strand break repair pathwayin mammalian cells. EMBO J 19: 3398–3407.

Johnson RD, Jasin M. 2001. Double-strand-break-inducedhomologous recombination in mammalian cells. Bio-chem Soc Trans 29: 196–201.

Johnson RD, Liu N, Jasin M. 1999. Mammalian XRCC2promotes the repair of DNA double-strand breaks byhomologous recombination. Nature 401: 397–399.

Johnson N, Johnson SF, Yao W, Li YC, Choi YE, BernhardyAJ, Wang Y, Capelletti M, Sarosiek KA, Moreau LA, et al.2013. Stabilization of mutant BRCA1 protein confersPARP inhibitor and platinum resistance. Proc Natl AcadSci 110: 17041–17046.

Jones S, Hruban RH, Kamiyama M, Borges M, Zhang X,Parsons DW, Lin JC, Palmisano E, Brune K, Jaffee EM, etal. 2009. Exomic sequencing identifies PALB2 as a pan-creatic cancer susceptibility gene. Science 324: 217.

Jonkers J, Meuwissen R, van der Gulden H, Peterse H, vander Valk M, Berns A. 2001. Synergistic tumor suppressoractivity of BRCA2 and p53 in a conditional mouse modelfor breast cancer. Nat Genet 29: 418–425.

Jonsson G, Staaf J, Vallon-Christersson J, Ringner M, HolmK, Hegardt C, Gunnarsson H, Fagerholm R, Strand C,Agnarsson BA, et al. 2010. Genomic subtypes of breastcancer identified by array-comparative genomic hybrid-ization display distinct molecular and clinical character-istics. Breast Cancer Res 12: R42.

Kakarougkas A, Ismail A, Katsuki Y, Freire R, Shibata A,Jeggo PA. 2013. Co-operation of BRCA1 and POH1 re-lieves the barriers posed by 53BP1 and RAP80 to resec-tion. Nucleic Acids Res 41: 10298–10311.

Kass EM, Jasin M. 2010. Collaboration and competitionbetween DNA double-strand break repair pathways.FEBS Lett 584: 3703–3708.

Kim H, Chen J, Yu X. 2007. Ubiquitin-binding proteinRAP80 mediates BRCA1-dependent DNA damage re-sponse. Science 316: 1202–1205.

King MC. 2014. “The race” to clone BRCA1. Science 343:1462–1465.

King MC, Marks JH, Mandell JB. 2003. Breast and ovariancancer risks due to inherited mutations in BRCA1 andBRCA2. Science 302: 643–646.

King TA, Li W, Brogi E, Yee CJ, Gemignani ML, Olvera N,Levine DA, Norton L, Robson ME, Offit K, et al. 2007.Heterogenic loss of the wild-type BRCA allele in humanbreast tumorigenesis. Ann Surg Oncol 14: 2510–2518.

Kojic M, Yang H, Kostrub CF, Pavletich NP, Holloman WK.2003. The BRCA2-interacting protein DSS1 is vital forDNA repair, recombination, and genome stability in Us-tilago maydis. Mol Cell 12: 1043–1049.

Kojic M, Zhou Q, Lisby M, Holloman WK. 2005. Brh2-Dss1interplay enables properly controlled recombination inUstilago maydis. Mol Cell Biol 25: 2547–2557.

Kraakman-van der Zwet M, Overkamp WJ, van Lange RE,Essers J, van Duijn-Goedhart A, Wiggers I, SwaminathanS, van Buul PP, Errami A, Tan RT, et al. 2002. Brca2(XRCC11) deficiency results in radioresistant DNA syn-thesis and a higher frequency of spontaneous deletions.Mol Cell Biol 22: 669–679.

Kristensen CN, Bystol KM, Li B, Serrano L, Brenneman MA.2010. Depletion of DSS1 protein disables homologousrecombinational repair in human cells. Mutation Res694: 60–64.

Krogan NJ, Lam MH, Fillingham J, Keogh MC, Gebbia M, LiJ, Datta N, Cagney G, Buratowski S, Emili A, et al. 2004.Proteasome involvement in the repair of DNA double-strand breaks. Mol Cell 16: 1027–1034.

Kurumizaka H, Ikawa S, Nakada M, Eda K, Kagawa W,Takata M, Takeda S, Yokoyama S, Shibata T. 2001. Ho-mologous-pairing activity of the human DNA-repairproteins Xrcc3.Rad51C. Proc Natl Acad Sci 98: 5538–5543.

Kurumizaka H, Ikawa S, Nakada M, Enomoto R, Kagawa W,Kinebuchi T, Yamazoe M, Yokoyama S, Shibata T. 2002.Homologous pairing and ring and filament structureformation activities of the human Xrcc2.Rad51D com-plex. J Biol Chem 277: 14315–14320.

Kurumizaka H, Enomoto R, Nakada M, Eda K, Yokoyama S,Shibata T. 2003. Region and amino acid residues requiredfor Rad51C binding in the human Xrcc3 protein. NucleicAcids Res 31: 4041–4050.

Kuznetsov S, Pellegrini M, Shuda K, Fernandez-Capetillo O,Liu Y, Martin BK, Burkett S, Southon E, Pati D, TessarolloL, et al. 2007. RAD51C deficiency in mice results in earlyprophase I arrest in males and sister chromatid separationat metaphase II in females. J Cell Biol 176: 581–592.

Kuznetsov SG, Liu P, Sharan SK. 2008. Mouse embryonicstem cell-based functional assay to evaluate mutations inBRCA2. Nat Med 14: 875–881.

Kuznetsov SG, Haines DC, Martin BK, Sharan SK. 2009.Loss of Rad51c leads to embryonic lethality and modu-lation of Trp53-dependent tumorigenesis in mice. CancerRes 69: 863–872.

� Lam I, Keeney S. 2015. Mechanism and regulation of mei-otic recombination initiation. Cold Spring Harb PerspectBiol 7: a016634.

Laufer M, Nandula SV, Modi AP, Wang S, Jasin M, MurtyVV, Ludwig T, Baer R. 2007. Structural requirements forthe BARD1 tumor suppressor in chromosomal stabilityand homology-directed DNA repair. J Biol Chem 282:34325–34333.

Lee Y, McKinnon PJ. 2002. DNA ligase IV suppresses me-dulloblastoma formation. Cancer Res 62: 6395–6399.





Lee MS, Green R, Marsillac SM, Coquelle N, Williams RS,Yeung T, Foo D, Hau DD, Hui B, Monteiro AN, et al.2010. Comprehensive analysis of missense variations inthe BRCT domain of BRCA1 by structural and functionalassays. Cancer Res 70: 4880–4890.

Levitus M, Waisfisz Q, Godthelp BC, de Vries Y, Hussain S,Wiegant WW, Elghalbzouri-Maghrani E, Steltenpool J,Rooimans MA, Pals G, et al. 2005. The DNA helicaseBRIP1 is defective in Fanconi anemia complementationgroup J. Nat Genet 37: 934–935.

Levran O, Attwooll C, Henry RT, Milton KL, Neveling K, RioP, Batish SD, Kalb R, Velleuer E, Barral S, et al. 2005. TheBRCA1-interacting helicase BRIP1 is deficient in Fanconianemia. Nat Genet 37: 931–933.

Li ML, Greenberg RA. 2012. Links between genome integ-rity and BRCA1 tumor suppression. Trends Biochem Sci37: 418–424.

Li J, Zou C, Bai Y, Wazer DE, Band V, Gao Q. 2006. DSS1 isrequired for the stability of BRCA2. Oncogene 25: 1186–1194.

Liang F, Han M, Romanienko PJ, Jasin M. 1998. Homology-directed repair is a major double-strand break repairpathway in mammalian cells. Proc Natl Acad Sci 95:5172–5177.

Lim DS, Hasty P. 1996. A mutation in mouse rad51 results inan early embryonic lethal that is suppressed by a muta-tion in p53. Mol Cell Biol 16: 7133–7143.

Lio YC, Mazin AV, Kowalczykowski SC, Chen DJ. 2003.Complex formation by the human Rad51B andRad51C DNA repair proteins and their activities in vitro.J Biol Chem 278: 2469–2478.

Litman R, Peng M, Jin Z, Zhang F, Zhang J, Powell S, An-dreassen PR, Cantor SB. 2005. BACH1 is critical for ho-mologous recombination and appears to be the Fanconianemia gene product FANCJ. Cancer Cell 8: 255–265.

Liu N, Lamerdin JE, Tebbs RS, Schild D, Tucker JD, ShenMR, Brookman KW, Siciliano MJ, Walter CA, Fan W, etal. 1998. XRCC2 and XRCC3, new human Rad51-familymembers, promote chromosome stability and protectagainst DNA cross-links and other damages. Mol Cell 1:783–793.

Liu N, Schild D, Thelen MP, Thompson LH. 2002. Involve-ment of Rad51C in two distinct protein complexes ofRad51 paralogs in human cells. Nucleic Acids Res 30:1009–1015.

Liu Y, Masson JY, Shah R, O’Regan P, West SC. 2004.RAD51C is required for Holliday junction processing inmammalian cells. Science 303: 243–246.

Liu J, Doty T, Gibson B, Heyer WD. 2010. Human BRCA2protein promotes RAD51 filament formation on RPA-covered single-stranded DNA. Nat Struct Mol Biol 17:1260–1262.

Lomonosov M, Anand S, Sangrithi M, Davies R, Venkitara-man AR. 2003. Stabilization of stalled DNA replicationforks by the BRCA2 breast cancer susceptibility protein.Genes Dev 17: 3017–3022.

London TB, Barber LJ, Mosedale G, Kelly GP, Balasubrama-nian S, Hickson ID, Boulton SJ, Hiom K. 2008. FANCJ isa structure-specific DNA helicase associated with themaintenance of genomic G/C tracts. J Biol Chem 283:36132–36139.

Long DT, Raschle M, Joukov V, Walter JC. 2011. Mechanismof RAD51-dependent DNA interstrand cross-link repair.Science 333: 84–87.

Loveday C, Turnbull C, Ramsay E, Hughes D, Ruark E,Frankum JR, Bowden G, Kalmyrzaev B, Warren-PerryM, Snape K, et al. 2011. Germline mutations inRAD51D confer susceptibility to ovarian cancer. Nat Ge-net 43: 879–882.

Loveday C, Turnbull C, Ruark E, Xicola RM, Ramsay E,Hughes D, Warren-Perry M, Snape K, Eccles D, EvansDG, et al. 2012. Germline RAD51C mutations confersusceptibility to ovarian cancer. Nat Genet 44: 475–476; author reply 476.

Ludwig T, Chapman DL, Papaioannou VE, Efstratiadis A.1997. Targeted mutations of breast cancer susceptibilitygene homologs in mice: Lethal phenotypes of Brca1,Brca2, Brca1/Brca2, Brca1/p53, and Brca2/p53 nullizy-gous embryos. Genes Dev 11: 1226–1241.

Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE,Norville JE, Church GM. 2013. RNA-guided human ge-nome engineering via Cas9. Science 339: 823–826.

Marston NJ, Richards WJ, Hughes D, Bertwistle D, MarshallCJ, Ashworth A. 1999. Interaction between the product ofthe breast cancer susceptibility gene BRCA2 and DSS1, aprotein functionally conserved from yeast to mammals.Mol Cell Biol 19: 4633–4642.

Masson JY, Tarsounas MC, Stasiak AZ, Stasiak A, Shah R,McIlwraith MJ, Benson FE, West SC. 2001. Identificationand purification of two distinct complexes containing thefive RAD51 paralogs. Genes Dev 15: 3296–3307.

McCabe N, Turner NC, Lord CJ, Kluzek K, Bialkowska A,Swift S, Giavara S, O’Connor MJ, Tutt AN, ZdzienickaMZ, et al. 2006. Deficiency in the repair of DNA dam-age by homologous recombination and sensitivity topoly(ADP-ribose) polymerase inhibition. Cancer Res66: 8109–8115.

McCarthy EE, Celebi JT, Baer R, Ludwig T. 2003. Loss ofBard1, the heterodimeric partner of the Brca1 tumorsuppressor, results in early embryonic lethality and chro-mosomal instability. Mol Cell Biol 23: 5056–5063.

� Mehta A, Haber JE. 2014. Sources of DNA double-strandbreaks and models of recombinational DNA repair.Cold Spring Harb Perspect Biol 6: a016428.

Meindl A, Hellebrand H, Wiek C, Erven V, WappenschmidtB, Niederacher D, Freund M, Lichtner P, Hartmann L,Schaal H, et al. 2010. Germline mutations in breast andovarian cancer pedigrees establish RAD51C as a humancancer susceptibility gene. Nat Genet 42: 410–414.

Metcalfe K, Lubinski J, Lynch HT, Ghadirian P, Foulkes WD,Kim-Sing C, Neuhausen S, Tung N, Rosen B, Gronwald J,et al. 2010. Family history of cancer and cancer risks inwomen with BRCA1 or BRCA2 mutations. J Natl CancerInst 102: 1874–1878.

Meyer S, Tischkowitz M, Chandler K, Gillespie A, Birch JM,Evans DG. 2014. Fanconi anaemia, BRCA2 mutationsand childhood cancer: A developmental perspectivefrom clinical and epidemiological observations with im-plications for genetic counselling. J Med Genet 51: 71–75.

Miki Y, Swensen J, Shattuck-Eidens D, Futreal PA, Harsh-man K, Tavtigian S, Liu Q, Cochran C, Bennett LM, DingW, et al. 1994. A strong candidate for the breast and

R. Prakash et al.




ovarian cancer susceptibility gene BRCA1. Science 266:66–71.

Miller KA, Sawicka D, Barsky D, Albala JS. 2004. Domainmapping of the Rad51 paralog protein complexes. Nu-cleic Acids Res 32: 169–178.

Mills KD, Ferguson DO, Essers J, Eckersdorff M, Kanaar R,Alt FW. 2004. Rad54 and DNA Ligase IV cooperate tomaintain mammalian chromatid stability. Genes Dev 18:1283–1292.

Min A, Im SA, Yoon YK, Song SH, Nam HJ, Hur HS, KimHP, Lee KH, Han SW, Oh DY, et al. 2013. RAD51C-defi-cient cancer cells are highly sensitive to the PARP inhib-itor olaparib. Mol Cancer Ther 12: 865–877.

Mizuta R, LaSalle JM, Cheng HL, Shinohara A, Ogawa H,Copeland N, Jenkins NA, Lalande M, Alt FW. 1997.RAB22 and RAB163/mouse BRCA2: Proteins that spe-cifically interact with the RAD51 protein. Proc Natl AcadSci 94: 6927–6932.

Moldovan GL, D’Andrea AD. 2009. How the Fanconi ane-mia pathway guards the genome. Annu Rev Genet 43:223–249.

� Morrical SW. 2015. DNA pairing and annealing processes inhomologous recombination and homology-directed re-pair. Cold Spring Harb Perspect Biol 7: a016444.

Morris JR, Solomon E. 2004. BRCA1: BARD1 induces theformation of conjugated ubiquitin structures, dependenton K6 of ubiquitin, in cells during DNA replication andrepair. Hum Mol Genet 13: 807–817.

Moynahan ME. 2002. The cancer connection: BRCA1 andBRCA2 tumor suppression in mice and humans. Onco-gene 21: 8994–9007.

Moynahan ME, Jasin M. 2010. Mitotic homologous recom-bination maintains genomic stability and suppresses tu-morigenesis. Nat Rev Mol Cell Biol 11: 196–207.

Moynahan ME, Chiu JW, Koller BH, Jasin M. 1999. Brca1controls homology-directed DNA repair. Mol Cell 4:511–518.

Moynahan ME, Cui TY, Jasin M. 2001a. Homology-directedDNA repair, mitomycin-c resistance, and chromosomestability is restored with correction of a Brca1 mutation.Cancer Res 61: 4842–4850.

Moynahan ME, Pierce AJ, Jasin M. 2001b. BRCA2 is re-quired for homology-directed repair of chromosomalbreaks. Mol Cell 7: 263–272.

Nakanishi K, Yang YG, Pierce AJ, Taniguchi T, Digweed M,D’Andrea AD, Wang ZQ, Jasin M. 2005. Human Fanconianemia monoubiquitination pathway promotes homol-ogous DNA repair. Proc Natl Acad Sci 102: 1110–1115.

Nakanishi K, Cavallo F, Perrouault L, Giovannangeli C,Moynahan ME, Barchi M, Brunet E, Jasin M. 2011. Ho-mology-directed Fanconi anemia pathway cross-link re-pair is dependent on DNA replication. Nat Struct MolBiol 18: 500–503.

Nicodeme F, Geffroy S, Conti M, Delobel B, Soenen V,Grardel N, Porte H, Copin MC, Lai JL, Andrieux J.2005. Familial occurrence of thymoma and auto-immune diseases with the constitutional translocationt(14;20)(q24.1;p12.3). Genes Chrom Cancer 44: 154–160.

Nishikawa H, Ooka S, Sato K, Arima K, Okamoto J, KlevitRE, Fukuda M, Ohta T. 2004. Mass spectrometric and

mutational analyses reveal Lys-6-linked polyubiquitinchains catalyzed by BRCA1-BARD1 ubiquitin ligase. JBiol Chem 279: 3916–3924.

Oliver AW, Swift S, Lord CJ, Ashworth A, Pearl LH. 2009.Structural basis for recruitment of BRCA2 by PALB2.EMBO Rep 10: 990–996.

O’Regan P, Wilson C, Townsend S, Thacker J. 2001. XRCC2is a nuclear RAD51-like protein required for damage-dependent RAD51 focus formation without the needfor ATP binding. J Biol Chem 276: 22148–22153.

Orii KE, Lee Y, Kondo N, McKinnon PJ. 2006. Selectiveutilization of nonhomologous end-joining and homolo-gous recombination DNA repair pathways during ner-vous system development. Proc Natl Acad Sci 103:10017–10022.

Ozcelik H, Schmocker B, Di Nicola N, Shi XH, Langer B,Moore M, Taylor BR, Narod SA, Darlington G, AndrulisIL, et al. 1997. Germline BRCA2 6174delT mutations inAshkenazi Jewish pancreatic cancer patients. Nat Genet16: 17–18.

Park DJ, Lesueur F, Nguyen-Dumont T, Pertesi M, Odefrey F,Hammet F, Neuhausen SL, John EM, Andrulis IL, TerryMB, et al. 2012. Rare mutations in XRCC2 increase therisk of breast cancer. Am J Hum Genet 90: 734–739.

Pellegrini L, Yu DS, Lo T, Anand S, Lee M, Blundell TL,Venkitaraman AR. 2002. Insights into DNA recombina-tion from the structure of a RAD51-BRCA2 complex.Nature 420: 287–293.

Pennington KP, Walsh T, Harrell MI, Lee MK, Pennil CC,Rendi MH, Thornton A, Norquist BM, Casadei S, NordAS, et al. 2014. Germline and somatic mutations in ho-mologous recombination genes predict platinum re-sponse and survival in ovarian, fallopian tube, and peri-toneal carcinomas. Clin Cancer Res 20: 764–775.

Pierce AJ, Johnson RD, Thompson LH, Jasin M. 1999.XRCC3 promotes homology-directed repair of DNAdamage in mammalian cells. Genes Dev 13: 2633–2638.

Pierce AJ, Hu P, Han M, Ellis N, Jasin M. 2001. Ku DNA end-binding protein modulates homologous repair of dou-ble-strand breaks in mammalian cells. Genes Dev 15:3237–3242.

Pittman DL, Schimenti JC. 2000. Midgestation lethality inmice deficient for the RecA-related gene, Rad51d/Rad51l3. Genesis 26: 167–173.

Pittman DL, Weinberg LR, Schimenti JC. 1998. Identifica-tion, characterization, and genetic mapping of Rad51d, anew mouse and human RAD51/RecA-related gene. Ge-nomics 49: 103–111.

Proia TA, Keller PJ, Gupta PB, Klebba I, Jones AD, Sedic M,Gilmore H, Tung N, Naber SP, Schnitt S, et al. 2011.Genetic predisposition directs breast cancer phenotypeby dictating progenitor cell fate. Cell Stem Cell 8: 149–163.

Rafnar T, Gudbjartsson DF, Sulem P, Jonasdottir A, Sigurds-son A, Jonasdottir A, Besenbacher S, Lundin P, Stacey SN,Gudmundsson J, et al. 2011. Mutations in BRIP1 conferhigh risk of ovarian cancer. Nat Genet 43: 1104–1107.

Rahman N, Seal S, Thompson D, Kelly P, Renwick A, ElliottA, Reid S, Spanova K, Barfoot R, Chagtai T, et al. 2007.PALB2, which encodes a BRCA2-interacting protein, is abreast cancer susceptibility gene. Nat Genet 39: 165–167.





Ratajska M, Antoszewska E, Piskorz A, Brozek I, Borg A,Kusmierek H, Biernat W, Limon J. 2012. Cancer predis-posing BARD1 mutations in breast-ovarian cancer fam-ilies. Breast Cancer Res Treat 131: 89–97.

� Reams AB, Roth JR. 2015. Mechanisms of gene duplica-tion and amplification. Cold Spring Harb Perspect Biol7: a016592.

Reczek CR, Szabolcs M, Stark JM, Ludwig T, Baer R. 2013.The interaction between CtIP and BRCA1 is not essentialfor resection-mediated DNA repair or tumor suppres-sion. J Cell Biol 201: 693–707.

Reid S, Schindler D, Hanenberg H, Barker K, Hanks S, KalbR, Neveling K, Kelly P, Seal S, Freund M, et al. 2007.Biallelic mutations in PALB2 cause Fanconi anemia sub-type FA-N and predispose to childhood cancer. Nat Genet39: 162–164.

Reid LJ, Shakya R, Modi AP, Lokshin M, Cheng JT, Jasin M,Baer R, Ludwig T. 2008. E3 ligase activity of BRCA1 is notessential for mammalian cell viability or homology-di-rected repair of double-strand DNA breaks. Proc NatlAcad Sci 105: 20876–20881.

Richardson C, Jasin M. 2000. Coupled homologous andnonhomologous repair of a double-strand break pre-serves genomic integrity in mammalian cells. Mol CellBiol 20: 9068–9075.

Rijkers T, Van Den Ouweland J, Morolli B, Rolink AG, Baar-ends WM, Van Sloun PP, Lohman PH, Pastink A. 1998.Targeted inactivation of mouse RAD52 reduces homolo-gous recombination but not resistance to ionizing radia-tion. Mol Cell Biol 18: 6423–6429.

Rodrigue A, Coulombe Y, Jacquet K, Gagne JP, Roques C,Gobeil S, Poirier G, Masson JY. 2013. The RAD51 para-logs ensure cellular protection against mitotic defects andaneuploidy. J Cell Sci 126: 348–359.

Rothkamm K, Kruger I, Thompson LH, Lobrich M. 2003.Pathways of DNA double-strand break repair during themammalian cell cycle. Mol Cell Biol 23: 5706–5715.

Rouet P, Smih F, Jasin M. 1994. Introduction of double-strand breaks into the genome of mouse cells by expres-sion of a rare-cutting endonuclease. Mol Cell Biol 14:8096–8106.

Ruffner H, Joazeiro CA, Hemmati D, Hunter T, Verma IM.2001. Cancer-predisposing mutations within the RINGdomain of BRCA1: Loss of ubiquitin protein ligase activ-ity and protection from radiation hypersensitivity. ProcNatl Acad Sci 98: 5134–5139.

Sabatier R, Adelaide J, Finetti P, Ferrari A, Huiart L, Sobol H,Chaffanet M, Birnbaum D, Bertucci F. 2010. BARD1 ho-mozygous deletion, a possible alternative to BRCA1 mu-tation in basal breast cancer. Genes Chrom Cancer 49:1143–1151.

Saeki H, Siaud N, Christ N, Wiegant WW, van Buul PP, HanM, Zdzienicka MZ, Stark JM, Jasin M. 2006. Suppressionof the DNA repair defects of BRCA2-deficient cells withheterologous protein fusions. Proc Natl Acad Sci 103:8768–8773.

Sartori AA, Lukas C, Coates J, Mistrik M, Fu S, Bartek J, BaerR, Lukas J, Jackson SP. 2007. Human CtIP promotes DNAend resection. Nature 450: 509–514.

Sawyer SL, Tian L, Kahkonen M, Schwartzentruber J,Kircher M, Majewski J, Dyment DA, Innes AM, BoycottKM, Moreau LA, et al. 2015. Biallelic mutations in

BRCA1 cause a new Fanconi anemia subtype. CancerDiscov 5: 135–142.

Schild D, Lio YC, Collins DW, Tsomondo T, Chen DJ. 2000.Evidence for simultaneous protein interactions betweenhuman Rad51 paralogs. J Biol Chem 275: 16443–16449.

Schlacher K, Christ N, Siaud N, Egashira A, Wu H, Jasin M.2011. Double-strand break repair-independent role forBRCA2 in blocking stalled replication fork degradationby MRE11. Cell 145: 529–542.

Schlacher K, Wu H, Jasin M. 2012. A distinct replication forkprotection pathway connects Fanconi anemia tumor sup-pressors to RAD51-BRCA1/2. Cancer Cell 22: 106–116.

Schoenmakers EF, Huysmans C, Van de Ven WJ. 1999. Al-lelic knockout of novel splice variants of human recom-bination repair gene RAD51B in t(12;14) uterine leio-myomas. Cancer Res 59: 19–23.

Scully R, Chen J, Plug A, Xiao Y, Weaver D, Feunteun J,Ashley T, Livingston DM. 1997. Association of BRCA1with Rad51 in mitotic and meiotic cells. Cell 88: 265–275.

Seal S, Thompson D, Renwick A, Elliott A, Kelly P, Barfoot R,Chagtai T, Jayatilake H, Ahmed M, Spanova K, et al. 2006.Truncating mutations in the Fanconi anemia J geneBRIP1 are low-penetrance breast cancer susceptibility al-leles. Nat Genet 38: 1239–1241.

Shakya R, Szabolcs M, McCarthy E, Ospina E, Basso K,Nandula S, Murty V, Baer R, Ludwig T. 2008. Thebasal-like mammary carcinomas induced by Brca1 orBard1 inactivation implicate the BRCA1/BARD1 heter-odimer in tumor suppression. Proc Natl Acad Sci 105:7040–7045.

Shakya R, Reid LJ, Reczek CR, Cole F, Egli D, Lin CS, deRooijDG, Hirsch S, Ravi K, Hicks JB, et al. 2011. BRCA1 tumorsuppression depends on BRCT phosphoprotein binding,but not its E3 ligase activity. Science 334: 525–528.

Shamseldin HE, Elfaki M, Alkuraya FS. 2012. Exome se-quencing reveals a novel Fanconi group defined byXRCC2 mutation. J Med Genet 49: 184–186.

Sharan SK, Morimatsu M, Albrecht U, Lim DS, Regel E,Dinh C, Sands A, Eichele G, Hasty P, Bradley A. 1997.Embryonic lethality and radiation hypersensitivity medi-ated by Rad51 in mice lacking Brca2. Nature 386: 804–810.

Shim KS, Schmutte C, Tombline G, Heinen CD, Fishel R.2004. hXRCC2 enhances ADP/ATP processing andstrand exchange by hRAD51. J Biol Chem 279: 30385–30394.

Shin DS, Pellegrini L, Daniels DS, Yelent B, Craig L, Bates D,Yu DS, Shivji MK, Hitomi C, Arvai AS, et al. 2003. Full-length archaeal Rad51 structure and mutants: Mecha-nisms for RAD51 assembly and control by BRCA2.EMBO J 22: 4566–4576.

Shu Z, Smith S, Wang L, Rice MC, Kmiec EB. 1999. Dis-ruption of mREC2/RAD51L1 in mice results in early em-bryonic lethality which can be partially rescued in ap532/2 background. Mol Cell Biol 19: 8686–8693.

Siaud N, Barbera MA, Egashira A, Lam I, Christ N,Schlacher K, Xia B, Jasin M. 2011. Plasticity of BRCA2function in homologous recombination: Genetic inter-actions of the PALB2 and DNA binding domains. PLoSGenet 7: e1002409.

R. Prakash et al.




Sigurdsson S, Van Komen S, Bussen W, Schild D, Albala JS,Sung P. 2001. Mediator function of the human Rad51B–Rad51C complex in Rad51/RPA-catalyzed DNA strandexchange. Genes Dev 15: 3308–3318.

Skoulidis F, Cassidy LD, Pisupati V, Jonasson JG, BjarnasonH, Eyfjord JE, Karreth FA, Lim M, Barber LM, Clatwor-thy SA, et al. 2010. Germline Brca2 heterozygosity pro-motes Kras(G12D)-driven carcinogenesis in a murinemodel of familial pancreatic cancer. Cancer Cell 18:499–509.

Smeenk G, de Groot AJ, Romeijn RJ, van Buul PP, Zdzie-nicka MZ, Mullenders LH, Pastink A, Godthelp BC.2010. Rad51C is essential for embryonic developmentand haploinsufficiency causes increased DNA damagesensitivity and genomic instability. Mutat Res 689: 50–58.

Smiraldo PG, Gruver AM, Osborn JC, Pittman DL. 2005.Extensive chromosomal instability in Rad51d-deficientmouse cells. Cancer Res 65: 2089–2096.

Sobhian B, Shao G, Lilli DR, Culhane AC, Moreau LA, Xia B,Livingston DM, Greenberg RA. 2007. RAP80 targetsBRCA1 to specific ubiquitin structures at DNA damagesites. Science 316: 1198–1202.

Somyajit K, Subramanya S, Nagaraju G. 2012. Distinct rolesof FANCO/RAD51C protein in DNA damage signalingand repair: Implications for Fanconi anemia and breastcancer susceptibility. J Biol Chem 287: 3366–3380.

Stark JM, Hu P, Pierce AJ, Moynahan ME, Ellis N, Jasin M.2002. ATP hydrolysis by mammalian RAD51 has a keyrole during homology-directed DNA repair. J Biol Chem277: 20185–20194.

Stark JM, Pierce AJ, Oh J, Pastink A, Jasin M. 2004. Geneticsteps of mammalian homologous repair with distinctmutagenic consequences. Mol Cell Biol 24: 9305–9316.

Suzuki A, de la Pompa JL, Hakem R, Elia A, Yoshida R, MoR, Nishina H, Chuang T, Wakeham A, Itie A, et al. 1997.Brca2 is required for embryonic cellular proliferation inthe mouse. Genes Dev 11: 1242–1252.

Sy SM, Huen MS, Chen J. 2009. PALB2 is an integral com-ponent of the BRCA complex required for homologousrecombination repair. Proc Natl Acad Sci 106: 7155–7160.

� Syeda AH, Hawkins M, McGlynn P. 2014. Recombinationand replication. Cold Spring Harb Perspect Biol 6:a016550.

� Symington LS. 2014. End resection at DNA double-strandbreaks: Mechanism and regulation. Cold Spring Harb Per-spect Biol 6: a016436.

Takata M, Sasaki MS, Tachiiri S, Fukushima T, Sonoda E,Schild D, Thompson LH, Takeda S. 2001. Chromosomeinstability and defective recombinational repair inknockout mutants of the five Rad51 paralogs. Mol CellBiol 21: 2858–2866.

Tang J, Cho NW, Cui G, Manion EM, Shanbhag NM, Bo-tuyan MV, Mer G, Greenberg RA. 2013. Acetylation limits53BP1 association with damaged chromatin to promotehomologous recombination. Nat Struct Mol Biol 20:317–325.

Tarsounas M, Munoz P, Claas A, Smiraldo PG, Pittman DL,Blasco MA, West SC. 2004. Telomere maintenance re-quires the RAD51D recombination/repair protein. Cell117: 337–347.

Tavtigian SV, Simard J, Rommens J, Couch F, Shattuck-Ei-dens D, Neuhausen S, Merajver S, Thorlacius S, Offit K,Stoppa-Lyonnet D, et al. 1996. The complete BRCA2 geneand mutations in chromosome 13q-linked kindreds. NatGenet 12: 333–337.

Tebbs RS, Zhao Y, Tucker JD, Scheerer JB, Siciliano MJ,Hwang M, Liu N, Legerski RJ, Thompson LH. 1995.Correction of chromosomal instability and sensitivi-ty to diverse mutagens by a cloned cDNA of theXRCC3 DNA repair gene. Proc Natl Acad Sci 92:6354–6358.

Thorslund T, Esashi F, West SC. 2007. Interactions betweenhuman BRCA2 protein and the meiosis-specific recom-binase DMC1. EMBO J 26: 2915–2922.

Thorslund T, McIlwraith MJ, Compton SA, Lekomtsev S,Petronczki M, Griffith JD, West SC. 2010. The breastcancer tumor suppressor BRCA2 promotes the specifictargeting of RAD51 to single-stranded DNA. Nat StructMol Biol 17: 1263–1265.

Tischkowitz M, Xia B. 2010. PALB2/FANCN: Recombiningcancer and Fanconi anemia. Cancer Res 70: 7353–7359.

Tischkowitz M, Xia B, Sabbaghian N, Reis-Filho JS, HamelN, Li G, van Beers EH, Li L, Khalil T, Quenneville LA, etal. 2007. Analysis of PALB2/FANCN-associated breastcancer families. Proc Natl Acad Sci 104: 6788–6793.

Towler WI, Zhang J, Ransburgh DJ, Toland AE, Ishioka C,Chiba N, Parvin JD. 2013. Analysis of BRCA1 variants indouble-strand break repair by homologous recombina-tion and single-strand annealing. Hum Mutat 34: 439–445.

Tsuzuki T, Fujii Y, Sakumi K, Tominaga Y, Nakao K, Seki-guchi M, Matsushiro A, Yoshimura Y, Morita T. 1996.Targeted disruption of the Rad51 gene leads to lethalityin embryonic mice. Proc Natl Acad Sci 93: 6236–6240.

Tutt A, Gabriel A, Bertwistle D, Connor F, Paterson H, Pea-cock J, Ross G, Ashworth A. 1999. Absence of Brca2 caus-es genome instability by chromosome breakage and lossassociated with centrosome amplification. Curr Biol 9:1107–1110.

Tutt A, Bertwistle D, Valentine J, Gabriel A, Swift S, Ross G,Griffin C, Thacker J, Ashworth A. 2001. Mutation inBrca2 stimulates error-prone homology-directed repairof DNA double-strand breaks occurring between repeat-ed sequences. EMBO J 20: 4704–4716.

Urbin SS, Elvers I, Hinz JM, Helleday T, Thompson LH.2012. Uncoupling of RAD51 focus formation and cellsurvival after replication fork stalling in RAD51D nullCHO cells. Environ Mol Mutagen 53: 114–124.

van Asperen CJ, Brohet RM, Meijers-Heijboer EJ, Hooger-brugge N, Verhoef S, Vasen HF, Ausems MG, Menko FH,Gomez Garcia EB, Klijn JG, et al. 2005. Cancer risks inBRCA2 families: Estimates for sites other than breast andovary. J Med Genet 42: 711–719.

Vaz F, Hanenberg H, Schuster B, Barker K, Wiek C, Erven V,Neveling K, Endt D, Kesterton I, Autore F, et al. 2010.Mutation of the RAD51C gene in a Fanconi anemia-like disorder. Nat Genet 42: 406–409.

Venkitaraman AR. 2014. Cancer suppression by the chro-mosome custodians, BRCA1 and BRCA2. Science 343:1470–1475.

Waddell N, Arnold J, Cocciardi S, da Silva L, Marsh A, RileyJ, Johnstone CN, Orloff M, Assie G, Eng C, et al. 2010.





Subtypes of familial breast tumours revealed by expres-sion and copy number profiling. Breast Canc Res Treat123: 661–677.

Walsh T, Casadei S, Lee MK, Pennil CC, Nord AS, ThorntonAM, Roeb W, Agnew KJ, Stray SM, Wickramanayake A, etal. 2011. Mutations in 12 genes for inherited ovarian,fallopian tube, and peritoneal carcinoma identified bymassively parallel sequencing. Proc Natl Acad Sci 108:18032–18037.

Wang B, Elledge SJ. 2007. Ubc13/Rnf8 ubiquitin ligasescontrol foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage. Proc NatlAcad Sci 104: 20759–20763.

Wang B, Matsuoka S, Ballif BA, Zhang D, Smogorzewska A,Gygi SP, Elledge SJ. 2007. Abraxas and RAP80 form aBRCA1 protein complex required for the DNA damageresponse. Science 316: 1194–1198.

Westermark UK, Reyngold M, Olshen AB, Baer R, Jasin M,Moynahan ME. 2003. BARD1 participates with BRCA1in homology-directed repair of chromosome breaks. MolCell Biol 23: 7926–7936.

Wiese C, Collins DW, Albala JS, Thompson LH, KronenbergA, Schild D. 2002. Interactions involving the Rad51 pa-ralogs Rad51C and XRCC3 in human cells. Nucleic AcidsRes 30: 1001–1008.

Wiese C, Hinz JM, Tebbs RS, Nham PB, Urbin SS, CollinsDW, Thompson LH, Schild D. 2006. Disparate require-ments for the Walker A and B ATPase motifs of humanRAD51D in homologous recombination. Nucleic AcidsRes 34: 2833–2843.

Williams RS, Glover JN. 2003. Structural consequences of acancer-causing BRCA1-BRCT missense mutation. J BiolChem 278: 2630–2635.

Williams RS, Chasman DI, Hau DD, Hui B, Lau AY, GloverJN. 2003. Detection of protein folding defects caused byBRCA1-BRCT truncation and missense mutations. J BiolChem 278: 53007–53016.

Wong AK, Pero R, Ormonde PA, Tavtigian SV, Bartel PL.1997. RAD51 interacts with the evolutionarily conservedBRC motifs in the human breast cancer susceptibilitygene brca2. J Biol Chem 272: 31941–31944.

Wong AK, Ormonde PA, Pero R, Chen Y, Lian L, Salada G,Berry S, Lawrence Q, Dayananth P, Ha P, et al. 1998.Characterization of a carboxy-terminal BRCA1 interact-ing protein. Oncogene 17: 2279–2285.

Wooster R, Bignell G, Lancaster J, Swift S, Seal S, Mangion J,Collins N, Gregory S, Gumbs C, Micklem G. 1995. Iden-tification of the breast cancer susceptibility gene BRCA2.Nature 378: 789–792.

Wu LC, Wang ZW, Tsan JT, Spillman MA, Phung A, Xu XL,Yang MC, Hwang LY, Bowcock AM, Baer R. 1996. Iden-tification of a RING protein that can interact in vivo withthe BRCA1 gene product. Nat Genet 14: 430–440.

Wu Y, Shin-ya K, Brosh RM Jr. 2008. FANCJ helicase defec-tive in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability. Mol CellBiol 28: 4116–4128.

Wu-Baer F, Lagrazon K, Yuan W, Baer R. 2003. The BRCA1/BARD1 heterodimer assembles polyubiquitin chainsthrough an unconventional linkage involving lysine res-idue K6 of ubiquitin. J Biol Chem 278: 34743–34746.

� Wyatt HDM, West SC. 2014. Holliday junction resolvases.Cold Spring Harb Perspect Biol 6: a023192.

Xia F, Taghian DG, DeFrank JS, Zeng ZC, Willers H, IliakisG, Powell SN. 2001. Deficiency of human BRCA2 leads toimpaired homologous recombination but maintainsnormal nonhomologous end joining. Proc Natl AcadSci 98: 8644–8649.

Xia B, Sheng Q, Nakanishi K, Ohashi A, Wu J, Christ N, LiuX, Jasin M, Couch FJ, Livingston DM. 2006. Control ofBRCA2 cellular and clinical functions by a nuclear part-ner, PALB2. Mol Cell 22: 719–729.

Xia B, Dorsman JC, Ameziane N, de Vries Y, Rooimans MA,Sheng Q, Pals G, Errami A, Gluckman E, Llera J, et al.2007. Fanconi anemia is associated with a defect in theBRCA2 partner PALB2. Nat Genet 39: 159–161.

Yamada NA, Hinz JM, Kopf VL, Segalle KD, Thompson LH.2004. XRCC3 ATPase activity is required for normalXRCC3-Rad51C complex dynamics and homologous re-combination. J Biol Chem 279: 23250–23254.

Yang H, Jeffrey PD, Miller J, Kinnucan E, Sun Y, ThomaNH, Zheng N, Chen PL, Lee WH, Pavletich NP. 2002.BRCA2 function in DNA binding and recombinationfrom a BRCA2-DSS1-ssDNA structure. Science 297:1837–1848.

Yang H, Li Q, Fan J, Holloman WK, Pavletich NP. 2005. TheBRCA2 homologue Brh2 nucleates RAD51 filament for-mation at a dsDNA-ssDNA junction. Nature 433: 653–657.

Yokoyama H, Kurumizaka H, Ikawa S, Yokoyama S, ShibataT. 2003. Holliday junction binding activity of the humanRad51B protein. J Biol Chem 278: 2767–2772.

Yokoyama H, Sarai N, Kagawa W, Enomoto R, Shibata T,Kurumizaka H, Yokoyama S. 2004. Preferential bind-ing to branched DNA strands and strand-annealingactivity of the human Rad51B, Rad51C, Rad51D,and Xrcc2 protein complex. Nucleic Acids Res 32:2556–2565.

Yonetani Y, Hochegger H, Sonoda E, Shinya S, Yoshikawa H,Takeda S, Yamazoe M. 2005. Differential and collabora-tive actions of Rad51 paralog proteins in cellular responseto DNA damage. Nucleic Acids Res 33: 4544–4552.

Yoshihara T, Ishida M, Kinomura A, Katsura M, TsurugaT, Tashiro S, Asahara T, Miyagawa K. 2004. XRCC3deficiency results in a defect in recombination and in-creased endoreduplication in human cells. EMBO J 23:670–680.

Yu X, Chen J. 2004. DNA damage-induced cell cycle check-point control requires CtIP, a phosphorylation-depen-dent binding partner of BRCA1 C-terminal domains.Mol Cell Biol 24: 9478–9486.

Yu X, Wu LC, Bowcock AM, Aronheim A, Baer R. 1998. TheC-terminal (BRCT) domains of BRCA1 interact in vivowith CtIP, a protein implicated in the CtBP pathway oftranscriptional repression. J Biol Chem 273: 25388–25392.

Yu DS, Sonoda E, Takeda S, Huang CL, Pellegrini L, BlundellTL, Venkitaraman AR. 2003a. Dynamic control of Rad51recombinase by self-association and interaction withBRCA2. Mol Cell 12: 1029–1041.

R. Prakash et al.




Yu X, Chini CC, He M, Mer G, Chen J. 2003b. The BRCTdomain is a phospho-protein binding domain. Science302: 639–642.

Yu X, Fu S, Lai M, Baer R, Chen J. 2006. BRCA1 ubiquiti-nates its phosphorylation-dependent binding partnerCtIP. Genes Dev 20: 1721–1726.

Yun MH, Hiom K. 2009. CtIP-BRCA1 modulates the choiceof DNA double-strand-break repair pathway throughoutthe cell cycle. Nature 459: 460–463.

� Zelensky A, Kanaar R, Wyman C. 2014. Mediators of ho-mologous DNA pairing. Cold Spring Harb Perspect Biol6: a016451.

Zhang F, Fan Q, Ren K, Andreassen PR. 2009a. PALB2functionally connects the breast cancer susceptibilityproteins BRCA1 and BRCA2. Mol Cancer Res 7: 1110–1118.

Zhang F, Ma J, Wu J, Ye L, Cai H, Xia B, Yu X. 2009b. PALB2links BRCA1 and BRCA2 in the DNA-damage response.Curr Biol 19: 524–529.

Zhao GY, Sonoda E, Barber LJ, Oka H, Murakawa Y, YamadaK, Ikura T, Wang X, Kobayashi M, Yamamoto K, et al.2007. A critical role for the ubiquitin-conjugating en-zyme Ubc13 in initiating homologous recombination.Mol Cell 25: 663–675.

Zhong Q, Chen CF, Li S, Chen Y, Wang CC, Xiao J, Chen PL,Sharp ZD, Lee WH. 1999. Association of BRCA1 with thehRad50-hMre11-p95 complex and the DNA damage re-sponse. Science 285: 747–750.

Zhou Y, Caron P, Legube G, Paull TT. 2013. Quantitation ofDNA double-strand break resection intermediates in hu-man cells. Nucleic Acids Res 42: e19.

Zhu Q, Pao GM, Huynh AM, Suh H, Tonnu N, NederlofPM, Gage FH, Verma IM. 2011. BRCA1 tumour suppres-sion occurs via heterochromatin-mediated silencing.Nature 477: 179–184.

Zimmermann M, Lottersberger F, Buonomo SB, Sfeir A, deLange T. 2013. 53BP1 regulates DSB repair using Rif1 tocontrol 50 end resection. Science 339: 700–704.





2015; doi: 10.1101/cshperspect.a016600Cold Spring Harb Perspect Biol Rohit Prakash, Yu Zhang, Weiran Feng and Maria Jasin BRCA2, and Associated ProteinsHomologous Recombination and Human Health: The Roles of BRCA1,

Subject Collection DNA Recombination

Meiotic Recombination: The Essence of HeredityNeil Hunter Recombinational DNA Repair

An Overview of the Molecular Mechanisms of

Stephen C. Kowalczykowski

MaintenanceRegulation of Recombination and Genomic

Wolf-Dietrich HeyerHomologs during MeiosisRecombination, Pairing, and Synapsis of

Denise Zickler and Nancy Kleckner

Chromatin RemodelingFlexibility, Impact of Histone Modifications, and Initiation of Meiotic Homologous Recombination:

Lóránt Székvölgyi, Kunihiro Ohta and Alain Nicolas

MeiosisDNA Strand Exchange and RecA Homologs in

M. Scott Brown and Douglas K. Bishop

Recombination InitiationMechanism and Regulation of Meiotic

Isabel Lam and Scott KeeneyAneuploid Oocytes and Trisomy BirthsMeiosis and Maternal Aging: Insights from

al.Mary Herbert, Dimitrios Kalleas, Daniel Cooney, et

ProteinsThe Roles of BRCA1, BRCA2, and Associated Homologous Recombination and Human Health:

Rohit Prakash, Yu Zhang, Weiran Feng, et al.

Homeologous RecombinationMismatch Repair during Homologous and

Maria Spies and Richard Fishel

Cell Biology of Mitotic RecombinationMichael Lisby and Rodney Rothstein Amplification

Mechanisms of Gene Duplication and

Andrew B. Reams and John R. Roth

Homology-Directed RepairHomologous Recombination and DNA-Pairing and Annealing Processes in

Scott W. Morrical

at Functional and Dysfunctional TelomeresThe Role of Double-Strand Break Repair Pathways

Ylli Doksani and Titia de Lange

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Mediators of Homologous DNA PairingAlex Zelensky, Roland Kanaar and Claire Wyman Recombination

Regulation of DNA Pairing in Homologous

Kwon, et al.James M. Daley, William A. Gaines, YoungHo

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